Substrates, devices, and methods for quantitative liquid crystal assays

ABSTRACT

The present invention relates to the field of molecular diagnostics, and in particular to diagnostics based on a liquid crystal assay format. In particular, the present invention provided improved substrates and methods of using liquid crystal assays for quantitating the amount of an analyte in a sample. The present invention also provides materials and methods for detecting non-specific binding of an analyte to a substrate by using a liquid crystal assay format.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 12/694,678, filed Jan. 27, 2010, which is a divisional of U.S.patent application Ser. No. 10/227,974, filed Aug. 26, 2002, now U.S.Pat. No. 7,666,661, issued Feb. 23, 2010, which claims priority toexpired U.S. Provisional Patent Application Ser. No. 60/315,203, filedAug. 27, 2001, all of which are herein incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to the field of molecular diagnostics, andin particular to diagnostics based on a liquid crystal assay format.

BACKGROUND OF THE INVENTION

The detection of pathogen, protein, and nucleic acid targets inbiological samples forms the basis of the multi-billion dollar in vitrodiagnostic industry. Detection of protein and nucleic acid targets canbe divided into diagnostic and research based markets. The diagnosticmarket includes the detection and identification of pathogens such asviruses and bacteria, the identification of various genetic markers, andthe identification of markers associated with the presence of tumors.The research market includes the genomics and proteomics industries,which require analytical, drug discovery, and high-throughput screeningtechnologies.

The ability to diagnose patients at the “point of care” is expected toyield major savings to the health care industry and improve theeffectiveness of treatment. For example, “point of care” testing is arequirement for the effective use of anti-influenza drugs such asRELENZA and TAMIFLU. This means that the diagnoses of influenza virusinfection must be made while the patient is in the doctor's office.Recent studies in the United States indicate that that when ZSTATFLU, arapid, influenza virus diagnostic assay was used at the point of care,healthcare costs were significantly reduced by elimination ofinappropriate treatment and the timely initiation of effective therapy.As another example, the advent of personalized medicine will requiregenetic screening of individuals at the point of care to determinewhether the individual is a candidate for particular treatmentstrategies or will have an adverse reaction to the preferred medication.

Currently used diagnostic assays include radioimmunoassay (RIA),enzyme-linked immunosorbent assay (ELISA), agglutination assays, surfaceplasmon resonance (SPR), and polymerized multilayer assemblies for thedetection of receptor-ligand interactions (Charych et al., Science261:585 (1993); Pan et al., Langmuir 13:1365 (1997)). However, most ofthese method requires expensive reagents (e.g., radioactively labeledantibodies or antigens), are not adaptable to microarray format (e.g.,agglutinations assays), or require expensive, laboratory based equipment(e.g., SPR).

Although many of the conventional assay methods described above workvery well to detect the presence of target species, they are expensiveand often require instrumentation and highly trained individuals, whichmakes them difficult to use routinely in the field. Thus, a need existsfor assay devices and systems which are easier to use and which allowfor evaluation of samples in remote locations.

SUMMARY OF THE INVENTION

The present invention relates to the field of molecular diagnostics, andin particular to diagnostics based on a liquid crystal assay format.Accordingly, in some embodiments, the present invention provides methodscomprising providing a sample suspected of containing an analyte and aliquid crystal assay device; adding the analyte to the liquid crystalassay device under conditions such that the presence of the analytecauses a detectable ordering of mesogens in the liquid crystal assaydevice; and quantitating the amount of the analyte in the sample basedon the detectable ordering of mesogens. The present invention is notlimited to any particular liquid crystal assay device. Indeed, the useof a variety of liquid crystal assay devices is contemplated, including,but not limited to, crystal assay device comprising a first substratehaving a surface, the surface comprising a recognition moiety; and amesogenic layer oriented on the surface. In some preferred embodiments,the liquid crystal assay device further comprises an interface betweenthe mesogenic layer and a member selected from the group consisting ofgases, liquids, solids, and combinations thereof. In other preferredembodiments, the recognition moiety is attached to the surface by aninteraction which is a member selected from the group consisting ofcovalent bonding, ionic bonding, chemisorption, physisorption, andcombinations thereof. In still other embodiments, the surface furthercomprises an organic layer. In further preferred embodiments, therecognition moiety is attached to the organic layer by an interactionwhich is a member selected from the group consisting of covalentbonding, ionic bonding, chemisorption, physisorption, and combinationsthereof. The present invention is not limited to any particularrecognition moiety. Indeed, the use of a variety of recognition moietiesis contemplated, including, but not limited to those selected from thegroup consisting of polynucleotides, antigen binding molecules, andpolypeptides. Likewise, the methods of the present invention are notlimited to the detection of any particular analyte. Indeed, thedetection of a variety of analytes is contemplated, including, but notlimited to those selected from the group consisting of polypeptides,polynucleotides, organic analytes, and pathogens.

In additional preferred embodiments, the mesogenic layer comprises apolymeric mesogen. The present invention is not limited to the use of aparticular mesogen. Indeed, the use of a variety of mesogens iscontemplated, including, but not limited to mesogens selected from thegroup consisting of 4-cyano-4′-pentylbiphenyl,N-(4-methoxybenzylidene)-4-butlyaniline and combinations thereof. Inother embodiments, the mesogenic layer comprises a lyotropic liquidcrystal. The present invention is not limited to any particular surface.Indeed, the present invention contemplates the use of a variety ofsurfaces, including, but not limited to, metal surfaces and polymericsurfaces. In some particularly preferred embodiments, the surface is ametal surface. The present invention is not limited to any particularmetal surface. Indeed, the use of a variety of metal surfaces iscontemplated, including, but not limited to, metal surfaces selectedfrom the group consisting of gold, platinum, palladium, copper, nickel,silver, and combinations thereof. The present invention is not limitedto any particular substrate. Indeed, the use of a variety of substratesis contemplated, including, but not limited to substrates selected fromthe group consisting of flexible substrates, rigid substrates, opticallyopaque substrates, optically transparent substrates, conductingsubstrates, semiconducting substrates, and combinations thereof. Instill further embodiments, the substrate is selected from the groupconsisting of inorganic crystals, inorganic glasses, inorganic oxides,metals, organic polymers, and combinations thereof. The presentinvention is not limited to any particular organic polymer. Indeed, theuse of a variety of organic polymers is contemplated including, but notlimited to, organic polymers selected from the group consisting ofpolyvinylidene fluoride, polydimethylsiloxane, polycarbonate,polystyrene, polyurethane, polyisocyanoacrylate, epoxy and combinationsthereof.

In some further preferred embodiments, the substrate is heterogenous.The present invention is not limited to any particular type ofheterogeneity. Indeed, the present invention contemplates that a varietyof heterogenous substrates may be utilized. In some preferredembodiments, the heterogeneity is a gradient of topography across thesurface. In some particularly preferred embodiments, a difference inliquid crystal orientation across the gradient of topography iscorrelated to the concentration of the analyte in the sample.

In other embodiments, the device further comprises a dichroic orfluorescent dye in the mesogenic layer. In still further embodiments,the method further comprises the step of measuring the amount of lighttransmitted by the device, wherein the amount of light transmitted isproportional to the amount of the analyte in the sample.

In still other embodiments, the quantitating step comprises illuminatingthe liquid crystal assay device with a specific wavelength of light todetermine the degree of disorder introduced into the liquid crystalassay device. In still further embodiments, the methods further comprisethe step of measuring the amount of light transmitted by the device,wherein the amount of light transmitted is proportional to the amount ofthe analyte in the sample. In some embodiments, the quantitating step isperformed with a plate reader. In further embodiments, the plate readeris utilized to detect the detectable ordering of mesogens, wherein thedetectable ordering of mesogens is accompanied by a change selected fromthe group the consisting of a change in fluorescence, transmittance,birefringence, and absorbance changes that accompany the reorientationof the liquid crystal.

In some preferred embodiments, the quantitating step is performed bymeasurement of the threshold electrical field required to change theordering of the mesogens. Accordingly, in other preferred embodiments,the liquid crystal assay device further comprises electrodes, whereinthe electrodes apply an electric field across the device.

In still other embodiments, the present invention provides systems fordetecting an analyte in a sample comprising at least one substratehaving a surface comprising recognition moieties; a mesogenic layeroriented on the surface; and electrodes configured to apply anelectrical field across the surface. In other embodiments, the systemfurther comprises an interface between the mesogenic layer and a memberselected from the group consisting of gases, liquids, solids, andcombinations thereof. In further embodiments, the recognition moiety isattached to the surface by an interaction which is a member selectedfrom the group consisting of covalent bonding, ionic bonding,chemisorption, physisorption, and combinations thereof. As described inmore detail above, the present invention is not limited to anyparticular organic layer, substrate, surface, recognition moiety,analyte, mesogen, or organic polymer.

In other embodiments, the present invention provides systems fordetecting an analyte in a sample comprising at least one substratehaving a surface comprising recognition moieties; and a mesogenic layeroriented on the surface, wherein the mesogenic layer comprises acompound selected from the group consisting of a dichroic dye and afluorescent compound. The present invention is not limited to anyparticular dichroic dye or fluorescent compound. Indeed, the use of avariety of dichroic dyes and fluorescent compounds is contemplated,including, but not limited to those selected from the group consistingof azobenzene, BTBP, polyazocompounds, anthraquinone, perylene dyes, andcombination thereof. In some preferred embodiments, the fluorescentcompound is BTBP. In other embodiments, the system further comprises aninterface between the mesogenic layer and a member selected from thegroup consisting of gases, liquids, solids, and combinations thereof. Infurther embodiments, the recognition moiety is attached to the surfaceby an interaction which is a member selected from the group consistingof covalent bonding, ionic bonding, chemisorption, physisorption, andcombinations thereof. As described in more detail above, the presentinvention is not limited to any particular organic layer, substrate,surface, recognition moiety, analyte, mesogen, or organic polymer.

In still further embodiments, the present invention provides systems fordetecting an analyte in a sample comprising at least one substratehaving a surface comprising recognition moieties, wherein the surface isheterogenous; and a mesogenic layer oriented on the surface. The presentinvention is not limited to any particular type of heterogeneity.Indeed, the present invention contemplates that a variety ofheterogenous substrates may be utilized. In some preferred embodiments,the heterogeneity is a gradient of topography across the surface. Insome particularly preferred embodiments, a difference in liquid crystalorientation across the gradient of topography is correlated to theconcentration of the analyte in the sample. In other embodiments, thesystem further comprises an interface between the mesogenic layer and amember selected from the group consisting of gases, liquids, solids, andcombinations thereof. In further embodiments, the recognition moiety isattached to the surface by an interaction which is a member selectedfrom the group consisting of covalent bonding, ionic bonding,chemisorption, physisorption, and combinations thereof. As described inmore detail above, the present invention is not limited to anyparticular organic layer, substrate, surface, recognition moiety,analyte, mesogen, or organic polymer.

In still other embodiments, the present invention provides methodscomprising providing a substrate having at least one surface and atleast one analyte; nonspecifically binding at least one analyte to thesubstrate; contacting the at least one surface with a mesogenic layer;and detecting binding of the at least one analyte to substrate, whereinthe binding causes a reorientation of the mesogenic layer that can bedetected. In some embodiments, the surface further comprises an organiclayer. As described in more detail above, the present invention is notlimited to any particular organic layer, substrate, surface, recognitionmoiety, analyte, mesogen, or organic polymer.

In further embodiments, the present invention provides methodscomprising providing a substrate having at least one surface; andnanoblasting the substrate under conditions such that the surfaceuniformly orients mesogens when the substrate is contacted with amesogenic layer. In some embodiments, the method further comprise thestep of attaching a recognition moiety to the substrate. In still otherembodiments, the methods further comprise the step of attaching anorganic layer to the substrate. In other embodiments, the system furthercomprises an interface between the mesogenic layer and a member selectedfrom the group consisting of gases, liquids, solids, and combinationsthereof. In further embodiments, the recognition moiety is attached tothe surface by an interaction which is a member selected from the groupconsisting of covalent bonding, ionic bonding, chemisorption,physisorption, and combinations thereof. As described in more detailabove, the present invention is not limited to any particular organiclayer, substrate, surface, recognition moiety, analyte, mesogen, ororganic polymer.

In still other embodiments, the present invention provides methodscomprising providing a substrate having at least one surface; andstretching the substrate under conditions such that the surfaceuniformly orients mesogens when the substrate is contacted with amesogenic layer. In some embodiments, the method further comprises thestep of attaching a recognition moiety to the substrate. In still otherembodiments, the methods further comprise the step of attaching anorganic layer to the substrate. In other embodiments, the system furthercomprises an interface between the mesogenic layer and a member selectedfrom the group consisting of gases, liquids, solids, and combinationsthereof. In further embodiments, the recognition moiety is attached tothe surface by an interaction which is a member selected from the groupconsisting of covalent bonding, ionic bonding, chemisorption,physisorption, and combinations thereof. As described in more detailabove, the present invention is not limited to any particular organiclayer, substrate, surface, recognition moiety, analyte, mesogen, ororganic polymer.

DESCRIPTION OF THE FIGURES

FIG. 1 is schematic depiction of a plate reading device of the presentinvention.

FIG. 2 is schematic depiction of a plate reading device of the presentinvention.

FIGS. 3 A, B, C and D shows disrupted liquid crystal orientation due tonon-specific adsorption of BSA. The optical textures shown in FIG. 3Bwere obtained after rotation of the cell shown in FIG. 3A by 45°.

FIGS. 4 A, B, C and D shows uniform, homeotropic liquid crystalorientation in the absence of BSA. The optical appearance shown in FIG.4B was obtained after rotation of the cell shown in FIG. 4A by 45°.

FIGS. 5 A, B, C, and D shows optical textures of 5CB (crossedpolarizers) sandwiched between non-stretched Parafilm and OTS-coatedglass microscope slides. The optical textures shown in FIG. 5B wereobtained after rotation of the cell shown in FIG. 5A by 45°.

FIGS. 6 A and B show optical textures of 5CB (crossed polarizers)sandwiched between stretched Parafilm and OTS-coated glass microscopeslides. The optical textures shown in FIG. 6B were obtained afterrotation of the cell shown in FIG. 5A by 45°.

FIG. 7 shows the effect of a rough microscope slide on opticalappearance. FIG. 7A shows uniform, aligned liquid crystal orientation.Optical textures of 5CB (cross polarizers) sandwiched between a roughglass microscope slide and a clean glass microscope slide. The opticalappearance shown in FIG. 7B was obtained after rotation of cell A by45°.

DEFINITIONS

As used herein, the term “immobilization” refers to the attachment orentrapment, either chemically or otherwise, of a material to anotherentity (e.g., a solid support) in a manner that restricts the movementof the material.

As used herein, the terms “material” and “materials” refer to, in theirbroadest sense, any composition of matter.

As used herein the term “polypeptide” is used in its broadest sense torefer to all molecules or molecular assemblies containing two or moreamino acids. Such molecules include, but are not limited to, proteins,peptides, enzymes, antibodies, receptors, lipoproteins, andglycoproteins.

As used herein the term “antigen binding protein” refers to aglycoprotein evoked in an animal by an immunogen (antigen) and toproteins derived from such glycoprotein (e.g., single chain antibodiesand F(ab′)2, Fab′ and Fab fragments). An antibody demonstratesspecificity to the immunogen, or, more specifically, to one or moreepitopes contained in the immunogen. Native antibody comprises at leasttwo light polypeptide chains and at least two heavy polypeptide chains.Each of the heavy and light polypeptide chains contains at the aminoterminal portion of the polypeptide chain a variable region (i.e., VHand VL respectively), which contains a binding domain that interactswith antigen. Each of the heavy and light polypeptide chains alsocomprises a constant region of the polypeptide chains (generally thecarboxy terminal portion) which may mediate the binding of theimmunoglobulin to host tissues or factors influencing various cells ofthe immune system, some phagocytic cells and the first component (Clq)of the classical complement system. The constant region of the lightchains is referred to as the “CL region,” and the constant region of theheavy chain is referred to as the “CH region.” The constant region ofthe heavy chain comprises a CH1 region, a CH2 region, and a CH3 region.A portion of the heavy chain between the CH₁ and CH2 regions is referredto as the hinge region (i.e., the “H region”). The constant region ofthe heavy chain of the cell surface form of an antibody furthercomprises a spacer-transmembranal region (M1) and a cytoplasmic region(M2) of the membrane carboxy terminus. The secreted form of an antibodygenerally lacks the M1 and M2 regions.

As used herein, the term “analytes” refers to any material that is to beanalyzed. Such materials can include, but are not limited to, ions,molecules, proteins, nucleic acids, antigens, bacteria, compounds,viruses, cells, antibodies, and cell parts.

As used herein, the term “selective binding” refers to the binding ofone material to another in a manner dependent upon the presence of aparticular molecular structure (i.e., specific binding). For example, areceptor will selectively bind ligands that contain the chemicalstructures complementary to the ligand binding site(s). This is incontrast to “non-selective binding,” whereby interactions are arbitraryand not based on structural compatibilities of the molecules.

As used herein, the term “conformational change” refers to thealteration of the molecular structure of a substance. It is intendedthat the term encompass the alteration of the structure of a singlemolecule or molecular aggregate (e.g., the change in structure of areceptor upon binding a ligand).

As used herein, the term “pathogen” refers to disease causing organisms,microorganisms, or agents including, but not limited to, viruses,bacteria, parasites (including, but not limited to, organisms within thephyla Protozoa, Platyhelminthes, Aschelminithes, Acanthocephala, andArthropoda), fungi, and prions.

As used herein, the term “bacteria” and “bacterium” refer to allprokaryotic organisms, including those within all of the phyla in theKingdom Procaryotae. It is intended that the term encompass allmicroorganisms considered to be bacteria including Mycoplasma,Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms ofbacteria are included within this definition including cocci, bacilli,spirochetes, spheroplasts, protoplasts, etc. “Gram negative” and “grampositive” refer to staining patterns obtained with the Gram-stainingprocess which is well known in the art (See e.g., Finegold and Martin,Diagnostic Microbiology, 6th Ed. (1982), C V Mosby St. Louis, pp 13-15).

As used herein, the term “polymerization” encompasses any process thatresults in the conversion of small molecular monomers into largermolecules consisting of repeated units. Typically, polymerizationinvolves chemical crosslinking of monomers to one another.

As used herein, the term “membrane receptors” refers to constituents ofmembranes that are capable of interacting with other molecules ormaterials. Such constituents can include, but are not limited to,proteins, lipids, carbohydrates, and combinations thereof.

As used herein, the term “volatile organic compound” or “VOC” refers toorganic compounds that are reactive (i.e., evaporate quickly, explosive,corrosive, etc.), and typically are hazardous to human health or theenvironment above certain concentrations. Examples of VOCs include, butare not limited to, alcohols, benzenes, toluenes, chloroforms, andcyclohexanes.

As used herein, the term “enzyme” refers to molecules or moleculeaggregates that are responsible for catalyzing chemical and biologicalreactions. Such molecules are typically proteins, but can also compriseshort peptides, RNAs, ribozymes, antibodies, and other molecules.

As used herein, the term “drug” refers to a substance or substances thatare used to diagnose, treat, or prevent diseases or conditions. Drugsact by altering the physiology of a living organism, tissue, cell, or invitro system that they are exposed to. It is intended that the termencompass antimicrobials, including, but not limited to, antibacterial,antifungal, and antiviral compounds. It is also intended that the termencompass antibiotics, including naturally occurring, synthetic, andcompounds produced by recombinant DNA technology.

As used herein, the term “carbohydrate” refers to a class of moleculesincluding, but not limited to, sugars, starches, cellulose, chitin,glycogen, and similar structures. Carbohydrates can also exist ascomponents of glycolipids and glycoproteins.

As used herein, the term “antigen” refers to any molecule or moleculargroup that is recognized by at least one antibody. By definition, anantigen must contain at least one epitope (i.e., the specificbiochemical unit capable of being recognized by the antibody). The term“immunogen” refers to any molecule, compound, or aggregate that inducesthe production of antibodies. By definition, an immunogen must containat least one epitope (i.e., the specific biochemical unit capable ofcausing an immune response).

As used herein, the term “chelating compound” refers to any compoundcomposed of or containing coordinate links that complete a closed ringstructure. The compounds can combine with metal ions, attached bycoordinate bonds to at least two of the nonmetal ions.

As used herein, the term “recognition moiety” refers to any molecule,molecular group, or molecular complex that is capable of recognizing(i.e., specifically interacting with) a molecule. For example, theligand binding site of a receptor would be considered a molecularrecognition complex.

As used herein, the terms “home testing” and “point of care testing”refer to testing that occurs outside of a laboratory environment. Suchtesting can occur indoors or outdoors at, for example, a privateresidence, a place of business, public or private land, in a vehicle, aswell as at the patient's bedside.

As used herein, the term “virus” refers to minute infectious agents,which with certain exceptions, are not observable by light microscopy,lack independent metabolism, and are able to replicate only within aliving host cell. The individual particles (i.e., virions) consist ofnucleic acid and a protein shell or coat; some virions also have a lipidcontaining membrane. The term “virus” encompasses all types of viruses,including animal, plant, phage, and other viruses.

As used herein, term “nanostructures” refers to microscopic structures,typically measured on a nanometer scale. Such structures include variousthree-dimensional assemblies, including, but not limited to, liposomes,films, multilayers, braided, lamellar, helical, tubular, and fiber-likeshapes, and combinations thereof. Such structures can, in someembodiments, exist as solvated polymers in aggregate forms such as rodsand coils. Such structures can also be formed from inorganic materials,such as prepared by the physical deposition of a gold film onto thesurface of a solid, proteins immobilized on surfaces that have beenmechanically rubbed, and polymeric materials that have been molded orimprinted with topography by using a silicon template prepared byelectron beam lithography.

As used the term “multilayer” refers to structures comprised of two ormore monolayers. The individual monolayers may chemically interact withone another (e.g., through covalent bonding, ionic interactions, van derWaals' interactions, hydrogen bonding, hydrophobic or hydrophilicassembly, and stearic hindrance) to produce a film with novel properties(i.e., properties that are different from those of the monolayersalone).

As used herein, the terms “self-assembling monomers” and “lipidmonomers” refer to molecules that spontaneously associate to formmolecular assemblies. In one sense, this can refer to surfactantmolecules that associate to form surfactant molecular assemblies. Theterm “self-assembling monomers” includes single molecules (e.g., asingle lipid molecule) and small molecular assemblies (e.g., polymerizedlipids), whereby the individual small molecular assemblies can befurther aggregated (e.g., assembled and polymerized) into largermolecular assemblies.

As used herein, the term “ligands” refers to any ion, molecule,molecular group, or other substance that binds to another entity to forma larger complex. Examples of ligands include, but are not limited to,peptides, carbohydrates, nucleic acids, antibodies, or any moleculesthat bind to receptors.

As used herein, the terms “organic matrix” and “biological matrix” referto collections of organic molecules that are assembled into a largermulti-molecular structure. Such structures can include, but are notlimited to, films, monolayers, and bilayers. As used herein, the term“organic monolayer” refers to a thin film comprised of a single layer ofcarbon-based molecules. In one embodiment, such monolayers can becomprised of polar molecules whereby the hydrophobic ends all line up atone side of the monolayer. The term “monolayer assemblies” refers tostructures comprised of monolayers. The term “organic polymetric matrix”refers to organic matrices whereby some or all of the molecularconstituents of the matrix are polymerized.

As used herein, the term “linker” or “spacer molecule” refers tomaterial that links one entity to another. In one sense, a molecule ormolecular group can be a linker that is covalent attached two or moreother molecules (e.g., linking a ligand to a self-assembling monomer).

As used herein, the term “bond” refers to the linkage between atoms inmolecules and between ions and molecules in crystals. The term “singlebond” refers to a bond with two electrons occupying the bonding orbital.Single bonds between atoms in molecular notations are represented by asingle line drawn between two atoms (e.g., C—C). The term “double bond”refers to a bond that shares two electron pairs. Double bonds arestronger than single bonds and are more reactive. The term “triple bond”refers to the sharing of three electron pairs. As used herein, the term“ene-yne” refers to alternating double and triple bonds. As used hereinthe terms “amine bond,” “thiol bond,” and “aldehyde bond” refer to anybond formed between an amine group (i.e., a chemical group derived fromammonia by replacement of one or more of its hydrogen atoms byhydrocarbon groups), a thiol group (i.e., sulfur analogs of alcohols),and an aldehyde group (i.e., the chemical group —CHO joined directlyonto another carbon atom), respectively, and another atom or molecule.

As used herein, the term “covalent bond” refers to the linkage of twoatoms by the sharing of two electrons, one contributed by each of theatoms.

As used herein, the term “spectrum” refers to the distribution of lightenergies arranged in order of wavelength.

As used the term “visible spectrum” refers to light radiation thatcontains wavelengths from approximately 360 nm to approximately 800 nm.

As used herein, the term “ultraviolet irradiation” refers to exposure toradiation with wavelengths less than that of visible light (i.e., lessthan approximately 360 nm) but greater than that of X-rays (i.e.,greater than approximately 0.1 nm). Ultraviolet radiation possessesgreater energy than visible light and is therefore, more effective atinducing photochemical reactions.

As used herein, the term “substrate” refers to a solid object or surfaceupon which another material is layered or attached. Solid supportsinclude, but are not limited to, glass, metals, gels, and filter paper,among others.

As used herein, the terms “array” and “patterned array” refer to anarrangement of elements (i.e., entities) into a material or device. Forexample, combining several types of ligand binding molecules (e.g.,antibodies or nucleic acids) into an analyte-detecting device, wouldconstitute an array.

As used herein, the term “badge” refers to any device that is portableand can be carried or worn by an individual working in an analytedetecting environment.

As used herein, the term “biological organisms” refers to anycarbon-based life forms.

As used herein, the term “in situ” refers to processes, events, objects,or information that are present or take place within the context oftheir natural environment.

As used herein, the term “sample” is used in its broadest sense. In onesense it can refer to a biopolymeric material. In another sense, it ismeant to include a specimen or culture obtained from any source, as wellas biological and environmental samples. Biological samples may beobtained from animals (including humans) and encompass fluids, solids,tissues, and gases. Biological samples include blood products, such asplasma, serum and the like. Environmental samples include environmentalmaterial such as surface matter, soil, water, crystals and industrialsamples. These examples are not to be construed as limiting the sampletypes applicable to the present invention.

As used herein, the term “liquid crystal” refers to a thermodynamicstable phase characterized by anisotropy of properties without theexistence of a three-dimensional crystal lattice, generally lying in thetemperature range between the solid and isotropic liquid phase.

As used herein, the term “mesogen” refers compounds that form liquidcrystals, and in particular rigid rodlike or disclike molecules whichare components of liquid crystalline materials.

As used herein, “thermotropic liquid crystal” refers to liquid crystalswhich result from the melting of mesogenic solids due to an increase intemperature. Both pure substances and mixtures form thermotropic liquidcrystals.

“Lyotropic,” as used herein, refers to molecules which form phases withorientational and/or positional order in a solvent. Lyotropic liquidcrystals can be formed using amphiphilic molecules (e.g., sodiumlaurate, phosphatidylethanolamine, lecithin). The solvent can be water.

As used herein, the term “heterogenous surface” refers to a surface thatorients liquid crystals in at least two separate planes or directions,such as across a gradient.

As used herein, “nematic” refers to liquid crystals in which the longaxes of the molecules remain substantially parallel, but the positionsof the centers of mass are randomly distributed. Nematic liquid crystalscan be substantially oriented by a nearby surface.

“Chiral nematic,” as used herein refers to liquid crystals in which themesogens are optically active. Instead of the director being heldlocally constant as is the case for nematics, the director rotates in ahelical fashion throughout the sample. Chiral nematic crystals show astrong optical activity which is much higher than can be explained onthe bases of the rotatory power of the individual mesogens. When lightequal in wavelength to the pitch of the director impinges on the liquidcrystal, the director acts like a diffraction grating, reflecting mostand sometimes all of the light incident on it. If white light isincident on such a material, only one color of light is reflected and itis circularly polarized. This phenomenon is known as selectivereflection and is responsible for the iridescent colors produced bychiral nematic crystals.

“Smectic,” as used herein refers to liquid crystals which aredistinguished from “nematics” by the presence of a greater degree ofpositional order in addition to orientational order; the molecules spendmore time in planes and layers than they do between these planes andlayers. “Polar smectic” layers occur when the mesogens have permanentdipole moments. In the smectic A2 phase, for example, successive layersshow anti ferroelectric order, with the direction of the permanentdipole alternating from layer to layer. If the molecule contains apermanent dipole moment transverse to the long molecular axis, then thechiral smectic phase is ferroelectric. A device utilizing this phase canbe intrinsically bistable.

“Frustrated phases,” as used herein, refers to another class of phasesformed by chiral molecules. These phases are not chiral, however, twistis introduced into the phase by an array of grain boundaries. A cubiclattice of defects (where the director is not defined) exist in acomplicated, orientationally ordered twisted structure. The distancebetween these defects is hundreds of nanometers, so these phases reflectlight just as crystals reflect x-rays.

“Discotic phases” are formed from molecules which are disc shaped ratherthan elongated. Usually these molecules have aromatic cores and sixlateral substituents. If the molecules are chiral or a chiral dopant isadded to a discotic liquid crystal, a chiral nematic discotic phase canform.

DESCRIPTION OF THE INVENTION

The present invention relates to the field of molecular diagnostics, andin particular to diagnostics based on a liquid crystal assay format.Liquid crystal-based assay systems (LC assays) are described in WO99/63329, which is herein incorporated by reference, and Gupta et al.,Science 279:2077-2080 (1998). Seung-Ryeol Kim, Rahul R. Shah, andNicholas L. Abbott; Orientations of Liquid Crystals on MechanicallyRubbed Films of Bovine Serum Albumin: A Possible Substrate forBiomolecular Assays Based on Liquid Crystals, Analytical Chemistry;2000; 72(19); 4646-4653; Justin J. Skaife and Nicholas L. Abbott;Quantitative Interpretation of the Optical Textures of Liquid CrystalsCaused by Specific Binding of Immunoglobulins to Surface-Bound Antigens,Langmuir; 2000; 16(7); 3529-3536; Vinay K. Gupta and Nicholas L. Abbott;Using proplets of Nematic Liquid Crystal To Probe the Microscopic andMesoscopic Structure of Organic Surfaces, Langmuir; 1999; 15(21);7213-7223.

The LC assays of the present inventions are useful for detecting andquantitating a wide variety of analytes, including, but not limited to,polypeptides, polynucleotides, viruses, microorganisms (bacteria,viruses, prions, fungi and mycoplasmas), and low molecular weightcompounds. It can also be used too discern subtle changes in an analytesuch as the activation state of a protein associated withphosphorylation. LC assays are used to directly detect specificmolecules and, in preferred embodiments, do not require labels,fluorescent dyes, colored substrates, or secondary antibodies.Furthermore, the LC assays of the present invention are readilyadaptable to multi-array formats that permit simultaneous detection ofmore than one target molecule, virus or micro-organism and appropriatecontrols. Adaptability to multi-array formats also makes the LC assaysof the present invention useful in high-throughput screeningapplications such as drug discovery. The LC assays of the presentinvention are also fast because the liquid crystals reorient in responseto alterations in a surface in seconds. Additionally, because the LCassays of the present invention do not, in preferred embodiments, needexpensive equipment to perform and interpret assay results, the LCassays are uniquely suited to on-site use and use in low-technologyenvironments.

In some embodiments of the present invention, but not all, the LC assayscomprise a substrate to which recognition moieties are attached,preferably via an organic layer on the substrate. In preferredembodiments, the substrate or organic layer serves to uniformly orientthe liquid crystal. In some preferred embodiments, the substrate surfaceis prepared by rubbing, nanoblasting (i.e., abrasion of a surface withsubmicron particles to create roughness), or oblique deposition of ametal. In some embodiments, the substrate so produced provides auniform, homogenous surface, while in other embodiments, the surface isheterogenous. In some particularly preferred embodiments, the substrateis patterned to allow quantification. When a target analyte binds to therecognition moiety, the orientation of the liquid crystal is disruptedat the discrete area of binding. According to the present invention, thedisruption of orientation can be detected by a variety of methods,including viewing with crossed polars, measuring the thresholdelectrical field required to change the orientation of the liquidcrystal, and viewing in the presence of dichroic agents. The liquidcrystals can be viewed using white light or using a specific wavelengthor combination of wavelengths of light.

Accordingly, the present invention provides improved substrates anddevices for LC assays, including quantitative LC assays. Forconvenience, the description of the present invention is divided intothe following sections: I. Substrates; II. Organic layers; III.Recognition moieties; IV. Mesogenic layers; V. Patterned liquidcrystals; VI. Analytes; VII. Compound libraries; VIII. Devices; and IX.Quantitation.

I. Substrates

Substrates that are useful in practicing the present invention can bemade of practically any physicochemically stable material. In apreferred embodiment, the substrate material is non-reactive towards theconstituents of the mesogenic layer. The substrates can be either rigidor flexible and can be either optically transparent or optically opaque.The substrates can be electrical insulators, conductors orsemiconductors. Further, the substrates can be substantially impermeableto liquids, vapors and/or gases or, alternatively, the substrates can bepermeable to one or more of these classes of materials. Exemplarysubstrate materials include, but are not limited to, inorganic crystals,inorganic glasses, inorganic oxides, metals, organic polymers andcombinations thereof.

A. Inorganic Crystal and Glasses

In some embodiments of the present invention, inorganic crystals andinorganic glasses are utilized as substrate materials (e.g., LiF, NaF,NaCl, KBr, KI, CaF₂, MgF₂, HgF₂, BN, AsS₃, ZnS, Si₃N₄ and the like). Thecrystals and glasses can be prepared by art standard techniques (See,e.g., Goodman, C. H. L., Crystal Growth Theory and Techniques, PlenumPress, New York 1974). Alternatively, the crystals can be purchasedcommercially (e.g., Fischer Scientific). The crystals can be the solecomponent of the substrate or they can be coated with one or moreadditional substrate components. Thus, it is within the scope of thepresent invention to utilize crystals coated with, for example one ormore metal films or a metal film and an organic polymer. Additionally, acrystal can constitute a portion of a substrate which contacts anotherportion of the substrate made of a different material, or a differentphysical form (e.g., a glass) of the same material. Other usefulsubstrate configurations utilizing inorganic crystals and/or glasseswill be apparent to those of skill in the art.

B. Inorganic Oxides

In other embodiments of the present invention, inorganic oxides areutilized as the substrate. Inorganic oxides of use in the presentinvention include, for example, Cs₂0, Mg(OH)₂, Ti0₂, Zr0₂, Ce0₂, Y₂0₃,Cr₂0₃, Fe₂0₃, NiO, ZnO, Al₂0₃, Si0₂ (glass), quartz, In₂0₃, Sn0₂, Pb0₂and the like. The inorganic oxides can be utilized in a variety ofphysical forms such as films, supported powders, glasses, crystals andthe like. A substrate can consist of a single inorganic oxide or acomposite of more than one inorganic oxide. For example, a composite ofinorganic oxides can have a layered structure (i.e., a second oxidedeposited on a first oxide) or two or more oxides can be arranged in acontiguous non-layered structure. In addition, one or more oxides can beadmixed as particles of various sizes and deposited on a support such asa glass or metal sheet. Further, a layer of one or more inorganic oxidescan be intercalated between two other substrate layers (e.g.,metal-oxide-metal, metal-oxide-crystal).

In a presently preferred embodiment, the substrate is a rigid structurethat is impermeable to liquids and gases. In this embodiment, thesubstrate consists of a glass plate onto which a metal, such as gold islayered by evaporative deposition. In a still further preferredembodiment, the substrate is a glass plate (Si0₂) onto which a firstmetal layer such as titanium has been layered. A layer of a second metalsuch as gold is then layered on top of the first metal layer.

C. Metals

In still further embodiments of the present invention, metals areutilized as substrates. The metal can be used as a crystal, a sheet or apowder. The metal can be deposited onto a backing by any method known tothose of skill in the art including, but not limited to, evaporativedeposition, sputtering, electroless deposition, electrolytic depositionand adsorption or deposition of preform particles of the metal includingmetallic nanoparticles.

Any metal that is chemically inert towards the mesogenic layer will beuseful as a substrate in the present invention. Metals that are reactiveor interactive towards the mesogenic layer will also be useful in thepresent invention. Metals that are presently preferred as substratesinclude, but are not limited to, gold, silver, platinum, palladium,nickel and copper. In one embodiment, more than one metal is used. Themore than one metal can be present as an alloy or they can be formedinto a layered “sandwich” structure, or they can be laterally adjacentto one another. In a preferred embodiment, the metal used for thesubstrate is gold. In a particularly preferred embodiment the metal usedis gold layered on titanium.

The metal layers can be either permeable or impermeable to materialssuch as liquids, solutions, vapors and gases.

D. Organic Polymers

In still other embodiments of the present invention, organic polymersare utilized as substrate materials. Organic polymers useful assubstrates in the present invention include polymers that are permeableto gases, liquids and molecules in solution. Other useful polymers arethose that are impermeable to one or more of these same classes ofcompounds.

Organic polymers that form useful substrates include, for example,polyalkenes (e.g., polyethylene, polyisobutene, polybutadiene),polyacrylics (e.g., polyacrylate, polymethyl methacrylate,polycyanoacrylate), polyvinyls (e.g., polyvinyl alcohol, polyvinylacetate, polyvinyl butyral, polyvinyl chloride), polystyrenes,polycarbonates, polyesters, polyurethanes, polyamides, polyimides,polysulfone, polysiloxanes, polyheterocycles, cellulose derivative(e.g., methyl cellulose, cellulose acetate, nitrocellulose),polysilanes, fluorinated polymers, epoxies, polyethers and phenolicresins (See, Cognard, J. ALIGNMENT OF NEMATIC LIQUID CRYSTALS AND THEIRMIXTURES, in Mol. Cryst. Liq. Cryst. 1:1-74 (1982)). Presently preferredorganic polymers include polydimethylsiloxane, polyethylene,polyacrylonitrile, cellulosic materials, polycarbonates and polyvinylpyridinium.

In a presently preferred embodiment, the substrate is permeable and itconsists of a layer of gold, or gold over titanium, which is depositedon a polymeric membrane, or other material, that is permeable toliquids, vapors and/or gases. The liquids and gases can be purecompounds (e.g., chloroform, carbon monoxide) or they can be compoundsthat are dispersed in other molecules (e.g., aqueous protein solutions,herbicides in air, alcoholic solutions of small organic molecules).Useful permeable membranes include, but are not limited to, flexiblecellulosic materials (e.g., regenerated cellulose dialysis membranes),rigid cellulosic materials (e.g., cellulose ester dialysis membranes),rigid polyvinylidene fluoride membranes, polydimethylsiloxane and tracketched polycarbonate membranes.

In a further preferred embodiment, the layer of gold on the permeablemembrane is itself permeable. In a still further preferred embodiment,the permeable gold layer has a thickness of about 70 Angstroms or less.

In those embodiments wherein the permeability of the substrate is not aconcern and a layer of a metal film is used, the film can be as thick asis necessary for a particular application. For example, if the film isused as an electrode, the film can be thicker than in an embodiment inwhich it is necessary for the film to be transparent or semi-transparentto light.

Thus, in a preferred embodiment, the film is of a thickness of fromabout 0.01 nanometer to about 1 micrometer. In a further preferredembodiment, the film is of a thickness of from about 5 nanometers toabout 100 nanometers. In yet a further preferred embodiment, the film isof a thickness of from about 10 nanometers to about 50 nanometers.

E. Substrate Surfaces

It is contemplated that the nature of the surface of the substrate has aprofound effect on the anchoring of the mesogenic layer that isassociated with the surface. The surface can be engineered by the use ofmechanical and/or chemical techniques. The surface of each of the aboveenumerated substrates can be substantially smooth. Alternatively, thesurface can be roughened or patterned by rubbing, etching, grooving,stretching, stressing, impacting, nanoblasting, oblique deposition orother similar techniques known to those of skill in the art. Ofparticular relevance is the texture of the surface that is in contactwith the mesogenic compounds.

Thus, in one preferred embodiment, the substrate is glass or an organicpolymer and the surface has been prepared by rubbing. Rubbing can beaccomplished using virtually any material including tissues, paper,fabrics, brushes, polishing paste, etc. In a preferred embodiment, therubbing is accomplished by use of a diamond rubbing paste. In anotherpreferred embodiment, the face of the substrate that contacts themesogenic compounds is a metal layer that has been obliquely depositedby evaporation. In a further preferred embodiment, the metal layer is agold layer.

In other embodiments of the present invention, anisotropic surfaces areprepared by nanoblasting a substrate with nanometer scale beads (e.g.,1-200 nm, preferably 50-100 nm) at a defined angle of incidence (e.g.,from about 5-85 degrees, preferably about 45 degrees). The nanoblastedsurface can be utilized as is or can be further modified, such as byobliquely depositing gold on the surface.

In still further embodiments, the ansiotropic surfaces of the devices ofthe present invention are prepared by stretching an appropriatesubstrate. For example, polymer substrates such as polystyrene can bestretched by heating to a temperature above the glass transitiontemperature of the substrate, applying a tensile force, and cooling to atemperature below the glass transition temperature before removing theforce.

In some embodiments, the present invention provides substrates withheterogenous features for use in the various devices and methods. Insome embodiments, the heterogenity is a uniform or non-uniform gradientin topography across the surface. For example, gold can be depositedonto a substrate at varying angles of incidence. Regions containing golddeposited at a near-normal angle of incidence will cause non-uniformanchoring of the liquid crystal, while areas in which the angle ofincidence was greater than 10 degrees will uniformally orient crystals.Alternatively, the heterogeneity may be the presence of two or moredistinct scales topography distributed uniformly across the substrate.It is contemplated that such substrates are useful for increasing thedynamic range of detection of analytes or for detecting the presence ofanalytes of a different size within a sample.

The substrate can also be patterned using techniques such asphotolithography (Kleinfield et al., J. Neurosci. 8:4098-120 (1998)),photoetching, chemical etching, microcontact printing (Kumar et al.,Langmuir 10:1498-511 (1994)), and chemical spotting.

The size and complexity of the pattern on the substrate is limited onlyby the resolution of the technique utilized and the purpose for whichthe pattern is intended. For example, using microcontact printing,features as small as 200 nm have been layered onto a substrate (See,Xia, Y.; Whitesides, G., J. Am. Chem. Soc. 117:3274-75 (1995)).Similarly, using photolithography, patterns with features as small as 1μm have been produced (See, Hickman et al., J. Vac. Sci. Technol.12:607-16 (1994)). Patterns which are useful in the present inventioninclude those which comprise features such as wells, enclosures,partitions, recesses, inlets, outlets, channels, troughs, diffractiongratings and the like.

In a presently preferred embodiment, the patterning is used to produce asubstrate having a plurality of adjacent wells, wherein each of thewells is isolated from the other wells by a raised wall or partition andthe wells do not fluidically communicate. Thus, an analyte, or othersubstance, placed in a particular well remains substantially confined tothat well. In another preferred embodiment, the patterning allows thecreation of channels through the device whereby an analyte can enterand/or exit the device.

The pattern can be printed directly onto the substrate or,alternatively, a “lift off” technique can be utilized. In the lift offtechnique, a patterned resist is laid onto the substrate, an organiclayer is laid down in those areas not covered by the resist and theresist is subsequently removed. Resists appropriate for use with thesubstrates of the present invention are known to those of skill in theart (See, e.g., Kleinfield et al., J. Neurosci. 8:4098-120 (1998)).Following removal of the photoresist, a second organic layer, having astructure different from the first organic layer, can be bonded to thesubstrate on those areas initially covered by the resist. Using thistechnique, substrates with patterns having regions of different chemicalcharacteristics can be produced. Thus, for example, a pattern having anarray of adjacent wells can be created by varying thehydrophobicity/hydrophilicity, charge and other chemical characteristicsof the pattern constituents. In one embodiment, hydrophilic compoundscan be confined to individual wells by patterning walls usinghydrophobic materials. Similarly, positively or negatively chargedcompounds can be confined to wells having walls made of compounds withcharges similar to those of the confined compounds. Similar substrateconfigurations are accessible through microprinting a layer with thedesired characteristics directly onto the substrate (See, Mrkish, M.;Whitesides, G. M., Ann. Rev. Biophys. Biomol. Struct. 25:55-78 (1996)).

In yet another preferred embodiment, the patterned substrate controlsthe anchoring alignment of the liquid crystal. In a particularlypreferred embodiment, the substrate is patterned with an organiccompound that forms a self-assembled monolayer. In this embodiment, theorganic layer controls the azimuthal orientation and/or polarorientation of a supported mesogenic layer.

F. Detection of Non-Specific Adsorption of Analytes

In some embodiments, substrates that uniformly orient mesogens areutilized to non-specifically bind analytes such as polypeptides orpolynucleotides. Accordingly, in some embodiments, the present inventionprovides methods for detecting molecules resolved by gel electophoresis,capillary electrophoreis, chromatography, and other separationtechnologies. Substrates suitable for detection of nonspecific bindinginclude rubbed PVDF membranes, rubbed nitrocellulose, and rubbedcellulose nitrate. In preferred embodiments, proteins or nucleic acidsfrom an electrophoretic gel are transferred to the substrate byapplication of an electric field in an appropriate buffer (e.g.,Western, Southern, or Northern blotting conditions). After transfer ofthe molecules to the surface, a mesogen layer and optionally a secondsubstrate that uniformly orients mesogens are applied as described inmore detail herein so that the non-specific binding can be detected. Inaddition to the analysis of resolved biomolecules, it is contemplatedthat analysis of non-specific binding is also useful quality control ofmanufactured biomolecules. In other embodiments of the invention, anobliquely deposited film of metal that supports an organic layer isused. A still preferred embodiment would be a self-assembled monolayerformed from an organosulfur compounds on the surface of a gold or silverfilm. In some cases, the self-assembled monolayer can be patterned withregions possessing different physical properties to affect theseparation of analytes from a mixture by their interaction with thesurface. A preferred pattern would be one in which their exists acontinuous gradient in properties across a surface.

II. Organic Layers

In addition to the ability of a substrate to anchor a mesogenic layer,an organic layer attached to the substrate is similarly able to providesuch anchoring. A wide range of organic layers can be used inconjunction with the present invention. These include, but are notlimited to, organic layers formed from organosulfur compounds (includingthiols and disulfides), organosilanes, amphiphilic molecules,cyclodextrins, polyols (e.g., poly(ethyleneglycol),poly(propyleneglycol), fullerenes, and biomolecules.

A. Anchoring

An organic layer that is bound to, supported on or adsorbed onto, thesurface of the substrate can anchor a mesogenic layer. As used herein,the term “anchoring” refers to the set of orientations adopted by themolecules in the mesogenic phase. The mesogenic layer will adoptparticular orientations while minimizing the free energy of theinterface between the organic layer and the mesogenic layer. Theorientation of the mesogenic layer is referred to as an “anchoringdirection.” A number of anchoring directions are possible.

It is contemplated that the particular anchoring direction adopted willdepend upon the nature of the mesogenic layer, the organic layer and thesubstrate. Anchoring directions of use in the present invention include,for example, conical anchoring, degenerate anchoring, homeotropicanchoring, multistable anchoring, planar anchoring and tilted anchoring.Planar anchoring and homeotropic anchoring are preferred with planaranchoring being most preferred.

The anchoring of mesogenic compounds by surfaces has been extensivelystudied for a large number of systems (See, for example, Jerome, Rep.Prog. Phys. 54:391-451 (1991)). The anchoring of a mesogenic substanceby a surface is specified, in general, by the orientation of thedirector of the bulk phase of the mesogenic layer. The orientation ofthe director, relative to the surface, is described by a polar angle(measured from the normal of the surface) and an azimuthal angle(measured in the plane of the surface).

Control of the anchoring of mesogens has been largely based on the useof organic surfaces prepared by coating surface-active molecules orpolymer films on inorganic (e.g., silicon oxide) substrates followed bysurface treatments such as rubbing. Other systems which have been founduseful include surfaces prepared through the reactions of organosilaneswith various substrates (See, for example, Yang et al., InMICROCHEMISTRY: SPECTROSCOPY AND CHEMISTRY IN SMALL DOMAINS; Masuhara etal., Eds.; North-Holland, Amsterdam, 1994; p. 441).

Molecularly designed surfaces formed by organic layers on a substratecan be used to control both the azimuthal and polar orientations of asupported mesogenic layer. SAMs can be patterned on a surface. Forexample, patterned organic layers made from CH₃(CH₂)₁₄SH andCH₃(CH₂)₁₅SH on obliquely deposited gold produce a supported mesogeniclayer which is twisted 90°. Other anchoring modes are readily accessibleby varying the chain length and the number of species of the organiclayer constituents (See, Gupta and Abbott, Science 276:1533-1536(1997)).

Transitions between anchoring modes have been obtained on a series oforganic layers by varying the structure of the organic layer. Thestructural features which have been found to affect the anchoring ofmesogenic compounds include, for example, the density of moleculeswithin the organic layer, the size and shape of the moleculesconstituting the organic layer and the number of individual layersmaking up the bulk organic layer.

The density of the organic layer on the substrate has been shown to havean effect on the mode of mesogen anchoring. For example, transitionsbetween homeotropic and degenerate anchorings have been obtained onsurfactant monolayers by varying the density of the monolayers (See,Proust et al., Solid State Commun. 11:1227-30 (1972)). Thus, it iswithin the scope of the present invention to tailor the anchoring modeof a mesogen by controlling the density of the organic layer on thesubstrate.

The molecular structure, size and shape of the individual moleculesmaking up the organic layer also affects the anchoring mode. Forexample, it has been demonstrated that varying the length of thealiphatic chains of surfactants on a substrate can also induce anchoringtransitions; with long chains, a homeotropic anchoring is obtained whilewith short chains, a conical anchoring is obtained with the tilt angle Oincreasing as the chain becomes shorter (See, e.g., Porte, J. Physique37:1245-52 (1976)). Additionally, recent reports have demonstrated thatthe polar angle of the mesogenic phase can be controlled by the choiceof the constituents of the organic layer. See, Gupta and Abbott,Langmuir 12:2587-2593 (1996). Thus, it is within the scope of thepresent invention to engineer the magnitude of the anchoring shift aswell as the type of shift by the judicious choice of organic layerconstituents.

Biomolecules can also be used as organic layers. (see Seung-Ryeol Kim,Rahul R. Shah, and Nicholas L. Abbott; Orientations of Liquid Crystalson Mechanically Rubbed Films of Bovine Serum Albumin: A PossibleSubstrate for Biomolecular Assays Based on Liquid Crystals, AnalyticalChemistry; 2000; 72(19); 4646-4653.). A preferred embodiment when usingbiomolecules as organic layers is based on the mechanical rubbing of theorganic layer with a fabric cloth following chemical immobilization ofthe organic layer on the surface of a substrate.

A wide variety of organic layers are useful in practicing the presentinvention. These organic layers can comprise monolayers, bilayers andmultilayers. Furthermore, the organic layers can be attached by covalentbonds, ionic bonds, physisorption, chemisorption and the like,including, but not limited to, hydrophobic interactions, hydrophilicinteractions, van der Waals interactions and the like.

In a presently preferred embodiment, organic layers which formselfassembled monolayers are used. The use of self-assembled monolayers(SAMs) formed from alkanethiols on thin, semitransparent films of goldin studies on the anchoring of liquid crystals on surfaces has beenreported (See, Drawhorn and Abbott, J. Phys. Chem. 45:16511 (1995)). Theprincipal result of that work was the demonstration that SAMs formedfrom n-alkanethiols with long (CH₃(CH₂)₁₅SH) and short (CH₃(CH₂)₄SH orCH₃(CH₂)₉SH) aliphatic chains can homeotropically anchor mesogens. Incontrast, single-component SAMs caused non-uniform, planar, or tiltedanchoring at room temperature.

In the discussion that follows, self-assembled monolayers are utilizedas an exemplary organic layer. This use is not intended to be limiting.It will be understood that the various configurations of theself-assembled monolayers and their methods of synthesis, bindingproperties and other characteristics are equally applicable to each ofthe organic layers of use in the present invention.

B. Self-Assembled Monolayers

Self-assembled monolayers are generally depicted as an assembly oforganized, closely packed linear molecules. There are two widely-usedmethods to deposit molecular monolayers on solid substrates:Langmuir-Blodgett transfer and self-assembly. Additional methods includetechniques such as depositing a vapor of the monolayer precursor onto asubstrate surface and the layer-by-layer deposition of polymers andpolyelectrolytes from solution (Guy Ladam, Pierre Schaaf, Frédéric J. G.Cuisinier, Gero Decher, and Jean-Claude Voegel; Protein Adsorption ontoAuto-Assembled Polyelectrolyte Films, Langmuir; 2001; 17(3); 878-882).

The composition of a layer of a SAM useful in the present invention canbe varied over a wide range of compound structures and molar ratios. Inone embodiment, the SAM is formed from only one compound. In a presentlypreferred embodiment, the SAM is formed from two or more components. Inanother preferred embodiment, when two or more components are used, onecomponent is a long-chain hydrocarbon having a chain length of between10 and 25 carbons and a second component is a short-chain hydrocarbonhaving a chain length of between 1 and 9 carbon atoms. In particularlypreferred embodiments, the SAM is formed from CH₃(CH₂)₁₅SH andCH₃(CH₂)₄SH or CH₃(CH₂)₁₅SH and CH₃(CH₂)₉SH. In any of theabove-described embodiments, the carbon chains can be functionalized atthe w-terminus (e.g., NH₂, COOH, OH, CN), at internal positions of thechain (e.g., aza, oxa, thia) or at both the w-terminus and internalpositions of the chain.

The mesogenic layer can be layered on top of one SAM layer or it can besandwiched between two SAM layers. In those embodiments in which themesogenic layer is sandwiched between two SAMs, a second substrate,optionally substantially identical in composition to that bearing theSAM can be layered on top of the mesogenic layer. Alternatively acompositionally different substrate can be layered on top of themesogenic layer. In a preferred embodiment, the second substrate ispermeable. In yet another preferred embodiment two substrates are used,but only one of the substrates has an attached organic layer.

When the mesogenic layer is sandwiched between two layers of SAMsseveral compositional permutations of the layers of SAMs are available.For example, in one embodiment, the first organic layer and the secondorganic layer have substantially identical compositions and both of theorganic layers bear an attached recognition moiety. A variation on thisembodiment utilizes first and second organic layers with substantiallysimilar compositions, wherein only one of the layers bears a recognitionmoiety.

In another embodiment, the first and second organic layers havesubstantially different compositions and only one of the organic layershas an attached recognition moiety. In a further embodiment, the firstorganic layer and said second organic layer have substantially differentcompositions and both of the organic layers have an attached recognitionmoiety.

In a presently preferred embodiment, the organic layers havesubstantially identical compositions and one or both of the organiclayers have attached thereto a recognition moiety.

A recognition moiety can be attached to the surface of a SAM by any of alarge number of art-known attachment methods. In one preferredembodiment, a reactive SAM component is attached to the substrate andthe recognition moiety is subsequently bound to the SAM component viathe reactive group on the component and a group of complementaryreactivity on the recognition moiety (See, e.g., Hegner et al. Biophys.J. 70:2052-2066 (1996)). In another preferred embodiment, therecognition moiety is attached to the SAM component prior toimmobilizing the SAM component on the substrate surface: the recognitionmoiety-SAM component cassette is then attached to the substrate. In astill further preferred embodiment, the recognition moiety is attachedto the substrate via a displacement reaction. In this embodiment, theSAM is preformed and then a fraction of the SAM components are displacedby a recognition moiety or a SAM component bearing a recognition moiety.

C. Functionalized SAMs

The discussion that follows focuses on the attachment of a reactive SAMcomponent to the substrate surface. This focus is for convenience onlyand one of skill in the art will understand that the discussion isequally applicable to embodiments in which the SAM component-recognitionmoiety is preformed prior to its attachment to the substrate. As usedherein, “reactive SAM components” refers to components which have afunctional group available for reaction with a recognition moiety orother species following the attachment of the component to thesubstrate.

Currently favored classes of reactions available with reactive SAMcomponents are those that proceed under relatively mild conditions.These include, but are not limited to nucleophilic substitutions (e.g.,reactions of amines and alcohols with acyl halides), electrophilicsubstitutions (e.g., enamine reactions) and additions to carbon-carbonand carbon-heteroatom multiple bonds (e.g., Michael reaction,Diels-Alder addition). These and other useful reactions are discussed inMarch, ADVANCED ORGANIC CHEMISTRY, Third Ed., John Wiley & Sons, NewYork, 1985.

According to the present invention, a substrate's surface isfunctionalized with SAM, components and other species by covalentlybinding a reactive SAM component to the substrate surface in such a wayas to derivatize the substrate surface with a plurality of availablereactive functional groups. Reactive groups which can be used inpracticing the present invention include, for example, amines, hydroxylgroups, carboxylic acids, carboxylic acid derivatives, alkenes,sulfhydryls, siloxanes, etc.

A wide variety of reaction types are available for the functionalizationof a substrate surface. For example, substrates constructed of a plasticsuch as polypropylene, can be surface derivatized by chromic acidoxidation, and subsequently converted to hydroxylated or aminomethylatedsurfaces. Substrates made from highly crosslinked divinylbenzene can besurface derivatized by chloromethylation and subsequent functional groupmanipulation. Additionally, functionalized substrates can be made frometched, reduced polytetrafluoroethylene.

When the substrates are constructed of a siliaceous material such asglass, the surface can be derivatized by reacting the surface Si—OH,Si0-H, and/or Si—Si groups with a functionalizing reagent. When thesubstrate is made of a metal film, the surface can be derivatized with amaterial displaying avidity for that metal.

In a preferred embodiment, wherein the substrates are made from glass,the covalent bonding of the reactive group to the glass surface isachieved by conversion of groups on the substrate's surface by a siliconmodifying reagent such as:

(RO)₃—Si—R¹—X¹  (1)

where R is an alkyl group, such as methyl or ethyl, R¹ is a linkinggroup between silicon and X and X is a reactive group or a protectedreactive group. The reactive group can also be a recognition moiety asdiscussed below. Silane derivatives having halogens or other leavinggroups beside the displayed alkoxy groups are also useful in the presentinvention.

A number of siloxane functionalizing reagents can be used, for example:

1. Hydroxyalkyl siloxanes (Silylate surface, functionalize withdiborane, and H₂0₂ to oxidize the alcohol)

a. allyl trichlorosilane→→3-hydroxypropyl

b. 7-oct-1-enyl trichlorosilane→→8-hydroxyoctyl

2. Diol (dihydroxyalkyl) siloxanes (silylate surface and hydrolyze todiol)

a. (glycidyl trimethoxysilane→→(2,3-dihydroxypropyloxy)propyl

3. Aminoalkyl siloxanes (amines requiring no intermediatefunctionalizing step).

a. 3-aminopropyl trimethoxysilane→aminopropyl

4. Dimeric secondary aminoalkyl siloxanes

a. bis(3-trimethoxysilylpropyl)amine→bis(silyloxylpropyl)amine.

It will be apparent to those of skill in the art that an array ofsimilarly useful functionalizing chemistries are available when SAMcomponents other than siloxanes are used. Thus, for example similarlyfunctionalized alkyl thiols can be attached to metal films andsubsequently reacted to produce the functional groups such as thoseexemplified above.

In another preferred embodiment, the substrate is at least partially ametal film, such as a gold film, and the reactive group is tethered tothe metal surface by an agent displaying avidity for that surface. In apresently preferred embodiment, the substrate is at least partially agold film and the group which reacts with the metal surface comprises athiol, sulfide or disulfide such as:

Y—S—R²—X²  (2)

R² is a linking group between sulfur and X² and X² is a reactive groupor a protected reactive group. X² can also be a recognition moiety asdiscussed below. Y is a member selected from the group consisting of H,R³ and R³—S—, wherein R² and R³ are independently selected. When R² andR³ are the same, symmetrical sulfides and disulfides result, and whenthey are different, asymmetrical sulfides and disulfides result.

A large number of functionalized thiols, sulfides and disulfides arecommercially available (Aldrich Chemical Co., St. Louis). Additionally,those of

skill in the art have available to them a manifold of synthetic routeswith which to produce additional such molecules. For example,amine-functionalized thiols can be produced from the correspondinghalo-amines, halo-carboxylic acids, etc. by reaction of these haloprecursors with sodium sulfhydride. See, e.g., Reid, ORGANIC CHEMISTRYof BIVALENT SULFUR, VOL 1, pp. 21-29, 32-35, vol. 5, pp. 27-34, ChemicalPublishing Co., New York, 1.958, 1963. Additionally, functionalizedsulfides can be prepared via alkylthio-de-halogenation with a mercaptansalt (See, Reid, ORGANIC CHEMISTRY OF BIVALENT SULFUR, vol. 2, pp.16-21, 24-29, vol. 3, pp. 11-14, Chemical Publishing Co., New York,1960). Other methods for producing compounds useful in practicing thepresent invention will be apparent to those of skill in the art.

In another preferred embodiment, the functionalizing reagent providesfor more than one reactive group per each reagent molecule. Usingreagents such as Compound 3, below, each reactive site on the substratesurface is, in essence, “amplified” to two or more functional groups:

(RO)₃—Si—R²—(X²)_(n)  (3)

where R is an alkyl group, such as methyl, R² is a linking group betweensilicon and X², X² is a reactive group or a protected reactive group andn is an integer between 2 and 50, and more preferably between 2 and 20.

Similar amplifying molecules are also of use in those embodimentswherein the substrate is at least partially a metal film. In theseembodiments the group that reacts with the metal surface comprises athiol, sulfide or disulfide such as in Formula (4):

Y—S—R²—(X²)_(n)  (4)

As discussed above, R² is a linking group between sulfur and X² and X²is a reactive group or a protected reactive group. X² can also be arecognition moiety. Y is a member selected from the group consisting ofH, R³ and R³—S—, wherein R² and R³ are independently selected.

R groups of use for R¹, R² and R³ in the above described embodiments ofthe present invention include, but are not limited to, alkyl,substituted alkyl, aryl, arylalkyl, substituted aryl, substitutedarylalkyl, acyl, halogen, hydroxy, amino, alkylamino, acylamino, alkoxy,acyloxy, aryloxy, aryloxyalkyl, mercapto, saturated cyclic hydrocarbon,unsaturated cyclic hydrocarbon, heteroaryl, heteroarylalkyl, substitutedheteroaryl, substituted heteroarylalkyl, heterocyclic, substitutedheterocyclic and heterocyclicalkyl groups.

In each of Formulae 1-4, above, each of R¹, R² and R³ are either stableor they can be cleaved by chemical or photochemical reactions. Forexample, R groups comprising ester or disulfide bonds can be cleaved byhydrolysis and reduction, respectively. Also within the scope of thepresent invention is the use of R groups, which are cleaved by lightsuch as, for example, nitrobenzyl derivatives, phenacyl groups, benzoinesters, etc. Other such cleaveable groups are well-known to those ofskill in the art.

In another preferred embodiment, the organosulfur compound is partiallyor entirely halogenated. An example of compounds useful in thisembodiment include:

X¹Q₂C(CQ¹ ₂)_(m)Z¹(CQ² ₂)_(n)SH  (5)

wherein, X¹ is a member selected from the group consisting of H, halogenreactive groups and protected reactive groups. Reactive groups can alsobe recognition moieties as discussed below. Q, Q¹ and Q² areindependently members selected from the group consisting of H andhalogen. Z¹ is a member selected from the group consisting of —CQ₂-,—CQ¹ ₂—, —CQ² ₂—, —O—, —S—, NR⁴—, —C(O)NR⁴ and R⁴NC(O0-, in which R⁴ isa member selected from the group consisting of H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups and mand n are independently a number between 0 and 40.

In yet another preferred embodiment, the organic layer comprises acompound according to Formula 5 above, in which Q, Q¹ and Q² areindependently members selected from the group consisting of H andfluorine. In a still further preferred embodiment, the organic layercomprises compounds having a structure according to Formulae (6) and(7):

CF₃(CF₂)_(m)Z¹(CH₂)_(n)SH  (6)

CF₃(CF₂)_(o)Z²(CH₂)_(p)SH  (7)

wherein, Z¹ and Z² are members independently selected from the groupconsisting of —CH₂—, —O—, —S—, NR⁴, —C(O)NR⁴ and R⁴NC(O)— in which R⁴ isa member selected from the group consisting of H, alkyl, substitutedalkyl, aryl, substituted aryl, heteroaryl and heterocyclic groups. In apresently preferred embodiment, the Z groups of adjacent moleculesparticipate in either an attractive (e.g., hydrogen bonding) orrepulsive (e.g., van der Waals) interaction.

In Formula 7, m is a number between 0 and 40, n is a number between 0and 40, o is a number between 0 and 40 and p is a number between 0 and40.

In a further preferred embodiment, the compounds of Formulae 6 and 7 areused in conjunction with an organosulfur compound, either halogentatedor unhalogenated, that bears a recognition moiety.

When the organic layer is formed from a halogenated organosulfurcompound, the organic layer can comprise a single halogenated compoundor more than one halogenated compound having different structures.Additionally, these layers can comprise a non-halogenated organosulfurcompound.

The reactive functional groups (X¹ and X²) are, for example:

(a) carboxyl groups and various derivatives thereof including, but notlimited to, N-hydroxysuccinimide esters, N-hydroxybenztriazole esters,acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl,alkenyl, alkynyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.

(c) haloalkyl groups wherein the halide can be later displaced with anucleophilic group such as, for example, an amine, a carboxylate anion,thiol anion, carbanion, or an alkoxide ion, thereby resulting in thecovalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido groups;

(e) aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, forexample, to form sulfonamides;

(g) thiol groups which can be converted to disulfides or reacted withacyl halides;

(h) amine or sulfhydryl groups which can be, for example, acylated oralkylated;

(i) alkenes which can undergo, for example, cycloadditions, acylation,Michael addition, etc; and

(j) epoxides which can react with, for example, amines and hydroxylcompounds.

The reactive moieties can also be recognition moieties. The nature ofthese groups is discussed in greater detail below.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reaction controlling theattachment of the functionalized SAM component onto the substrate'ssurface. Alternatively, the reactive functional group can be protectedfrom participating in the reaction by the presence of a protectinggroup. Those of skill in the art will understand how to protect aparticular functional group from interfering with a chosen set ofreaction conditions. For examples of useful protecting groups, seeGreene et al., PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, John Wiley &Sons, New York, 1991.

In a preferred embodiment, the SAM component bearing the recognitionmoiety is attached directly and essentially irreversibly via a “stablebond” to the surface of the substrate. A “stable bond”, as used herein,is a bond which maintains its chemical integrity over a wide range ofconditions (e.g., amide, carbamate, carbon-carbon, ether, etc.). Inanother preferred embodiment, the SAM component bearing the recognitionmoiety is attached to the substrate surface by a “cleaveable bond”. A“cleaveable bond”, as used herein, is a bond that is designed to undergoscission under conditions that do not degrade other bonds in therecognition moiety-analyte complex. Cleaveable bonds include, but arenot limited to, disulfide, imine, carbonate and ester bonds.

In certain embodiments, it is advantageous to have the recognitionmoiety attached to a SAM component having a structure that is differentthan that of the constituents of the bulk SAM. In this embodiment, thegroup to which the recognition moiety is bound is referred to as a“spacer arm” or “spacer.” Using such spacer arms, the properties of theSAM adjacent to the recognition moiety can be controlled. Propertiesthat are usefully controlled include, for example, hydrophobicity,hydrophilicity, surface-activity and the distance of the recognitionmoiety from the plane of the substrate and/or the SAM. For example, in aSAM composed of alkanethiols, the recognition moiety can be attached tothe substrate or the surface of the SAM via an amine terminatedpoly(ethyleneglycol). Numerous other combinations of spacer arms andSAMs are accessible to those of skill in the art.

The hydrophilicity of the substrate surface can be enhanced by reactionwith polar molecules such as amine-, hydroxyl- andpolyhydroxylcontaining molecules. Representative examples include, butare not limited to, polylysine, polyethyleneimine, poly(ethyleneglycol)and poly(propyleneglycol). Suitable functionalization chemistries andstrategies for these compounds are known in the art (See, for example,Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol. 469, American Chemical Society, Washington, D.C.1991).

The hydrophobicity of the substrate surface can be modulated by using ahydrophobic spacer arm such as, for example, long chain diamines,longchain thiols, α, ω-amino acids, etc. Representative hydrophobicspacers include, but are not limited to, 1,6-hexanediamine,1,8-octanediamine, 6-aminohexanoic acid and 8-aminooctanoic acid.

The substrate surface can also be made surface-active by attaching tothe substrate surface a spacer that has surfactant properties. Compoundsuseful for this purpose include, for example, aminated or hydroxylateddetergent molecules such as, for example, 1-aminododecanoic acid.

In another embodiment, the spacer serves to distance the recognitionmoiety from the substrate or SAM. Spacers with this characteristic haveseveral uses. For example, a recognition moiety held too closely to thesubstrate or SAM surface may not react with incoming analyte, or it mayreact unacceptably slowly. When an analyte is itself stericallydemanding, the reaction leading to recognition moiety-analyte complexformation can be undesirably slowed, or not occur at all, due to themonolithic substrate hindering the approach of the two components.

In another embodiment, the physicochemical characteristics (e.g.,hydrophobicity, hydrophilicity, surface activity, conformation) of thesubstrate surface and/or SAM are altered by attaching a monovalentmoiety which is different in composition than the constituents of thebulk SAM and which does not bear a recognition moiety. As used herein,“monovalent moiety” refers to organic molecules with only one reactivefunctional group. This functional group attaches the molecule to thesubstrate. “Monovalent moieties” are to be contrasted with thebifunctional “spacer” groups described above. Such monovalent groups areused to modify the hydrophilicity, hydrophobicity, bindingcharacteristics, etc. of the substrate surface. Examples of groupsuseful for this purpose include long chain alcohols, amines, fattyacids, fatty acid derivatives, poly(ethyleneglycol) monomethyl ethers,etc.

When two or more structurally distinct moieties are used as componentsof the SAMs, the components can be contacted with the substrate as amixture of SAM components or, alternatively, the components can be addedindividually. In those embodiments in which the SAM components are addedas a mixture, the mole ratio of a mixture of the components in solutionresults in the same ratio in the mixed SAM. Depending on the manner inwhich the SAM is assembled, the two components do not phase segregateinto islands (See, Bain and Whitesides, J. Am. Chem. Soc. 111:7164(1989)). This feature of SAMs can be used to immobilize recognitionmoieties or bulky modifying groups in such a manner that certaininteractions, such as steric hindrance, between these molecules isminimized.

The individual components of the SAMs can also be bound to the substratein a sequential manner. Thus, in one embodiment, a first SAM componentis attached to the substrate's surface by “underlabeling” the surfacefunctional groups with less than a stoichiometric equivalent of thefirst component. The first component can be a SAM component liked to aterminal reactive group or recognition group, a spacer arm or amonovalent moiety. Subsequently, the second component is contacted withthe substrate. This second component can either be added instoichiometric equivalence, stoichiometric excess or can again be usedto underlabel to leave sites open for a third component.

D. Detection of Non-Specific Adsorption of Analytes

In some embodiments, substrates prepared with an organic layer areutilized to non-specifically bind analytes such as polypeptides orpolynucleotides. Accordingly, in some embodiments, the present inventionprovides methods for detecting molecules resolved by gel electophoresis,capillary electrophoreis, chromatography, and other separationtechnologies. In some embodiments, the surface of the substrate iscoated with a monolayer that possesses a property useful fornon-specific adsorption of molecules or particular classes of molecules.For example, in some embodiments, a gold surface is coated with ahydrophobic monolayer (e.g., formed from hexadecanethiol) and a samplecontaining proteins is contacted with the hydrophobic monolayer underconditions such that the proteins in the sample associate with thehydrophobic monolayer. In preferred embodiments, proteins from anelectrophoretic gel are transferred to the hydrophobic surface byapplication of an electric field in an appropriate buffer (e.g., Westernblotting conditions). In other embodiments, the gold surface is coated apositively charged monolayer (e.g., formed from HS(CH₂)₈N⁺(CH₃)₃) thatbinds negatively charged polynucleotides (e.g., DNA or RNA). In stillfurther embodiments, the surface is prepared with patterned monolayerswith different functionalities (e.g., positive or negative charge wherethe negative charged regions are formed using SH(CH₂)₂SO₃ ⁻) so thatmolecules with different properties (e.g., isolectric point at a givenpH) bind to different areas of the surface. After transfer of themolecules to the surface, a mesogen layer and optionally a secondsubstrate that uniformly orients mesogens are applied as described inmore detail herein so that the non-specific binding can be detected.Mixed monolayers formed from positively charged, negatively charged andelectrically neutral species can be used to tune the properties of thesurface via variation of the composition of the mixed monolayer. Thesemixed monolayers can be prepared by co-adsorption, sequential adsorptionor displacement on the surface.

III. Recognition Moieties

In some embodiments of the present invention, a “recognition moiety”attached to or associated with the substrate is utilized to bind to orotherwise interact with another molecule or molecules (e.g., analytes).For example, in some embodiments, recognition moieties are attached toeither w-functionalized spacer arms or w-functionalized SAM components,which are in turn attached to or associated with the substrate.Furthermore, a recognition moiety can be presented by a polymer surface(e.g., a rubbed polymer surface).

In some preferred embodiments, the recognition moiety comprises anorganic functional group. In presently preferred embodiments, theorganic functional group is a member selected from the group consistingof amines, carboxylic acids, drugs, chelating agents, crown ethers,cyclodextrins or a combination thereof.

In another preferred embodiment, the recognition moiety is abiomolecule. In still further preferred embodiments, the biomolecule isa protein, antigen binding protein, peptide, nucleic acid (e.g., singlenucleotides or nucleosides, oligonucleotides, polynucleotides andsingle- and higher-stranded nucleic acids) or a combination thereof. Ina presently preferred embodiment, the recognition moiety is biotin. Insome embodiments of the present invention, the recognition moiety is anantigen binding protein. Such antigen binding proteins include, but arenot limited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, and Fab expression libraries.

Various procedures known in the art may be used for the production ofpolyclonal antibodies. For the production of antibody, various hostanimals, including but not limited to rabbits, mice, rats, sheep, goats,etc., can be immunized by injection with the peptide corresponding to anepitope. In a preferred embodiment, the peptide is conjugated to animmunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels (e.g., aluminum hydroxide), surface activesubstances (e.g., lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and Corynebacterium parvum).

For preparation of monoclonal antibodies, it is contemplated that anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture will find use with the presentinvention (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Theseinclude but are not limited to the hybridoma technique originallydeveloped by Köhler and Milstein (Köhler and Milstein, Nature256:495-497 [1975]), as well as the trioma technique, the human B-cellhybridoma technique (See e.g., Kozbor et al., Immunol. Tod., 4:72[1983]), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 [1985]).

In addition, it is contemplated that techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) will find use in producing specific singlechain antibodies that serve as recognition moieties. Furthermore, it iscontemplated that any technique suitable for producing antibodyfragments will find use in generating antibody fragments that are usefulrecognition moieties. For example, such fragments include but are notlimited to: F(ab′)2 fragment that can be produced by pepsin digestion ofthe antibody molecule; Fab′ fragments that can be generated by reducingthe disulfide bridges of the F(ab′)2 fragment, and Fab fragments thatcan be generated by treating the antibody molecule with papain and areducing agent. In still further embodiments, the recognition moietycomprises a phage displaying an antigen binding protein.

In some embodiments where the recognition moiety is a polynucleotide orpolypeptide, a plurality of recognition moieties are arrayed on thesubstrates using photo activated chemistry, microcontact printing, andink-jet printing. In particularly preferred embodiments,photolithography is utilized (See e.g., U.S. Pat. Nos. 6,045,996;5,925,525; and 5,858,659; each of which is herein incorporated byreference). Using a series of photolithographic masks to definesubstrate exposure sites, followed by specific chemical synthesis steps,the process constructs high-density arrays of oligonucleotides, witheach probe in a predefined position in the array. Multiple probe arraysare synthesized simultaneously on, for example, a large glass wafer. Thewafers are then diced, and individual probe arrays are packaged ininjection-molded plastic cartridges, which protect them from theenvironment and serve as chambers for hybridization.

In other embodiments, nucleic acid recognition moieties areelectronically captured on a suitable substrate (See e.g., U.S. Pat.Nos. 6,017,696; 6,068,818; and 6,051,380; each of which are hereinincorporated by reference). Through the use of microelectronics, thistechnology enables the active movement and concentration of chargedmolecules to and from designated test sites on its semiconductormicrochip. DNA capture probes unique to a given target areelectronically placed at, or “addressed” to, specific sites on themicrochip. Since DNA has a strong negative charge, it can beelectronically moved to an area of positive charge.

In still further embodiments, recognition moieties are arrayed on asuitable substrate by utilizing differences in surface tension (Seee.g., U.S. Pat. Nos. 6,001,311; 5,985,551; and 5,474,796; each of whichis herein incorporated by reference). This technology is based on thefact that fluids can be segregated on a flat surface by differences insurface tension that have been imparted by chemical coatings. Once sosegregated, oligonucleotide probes are synthesized directly on the chipby ink-jet printing of reagents. The array with its reaction sitesdefined by surface tension is mounted on a X/Y translation stage under aset of four piezoelectric nozzles, one for each of the four standard DNAbases. The translation stage moves along each of the rows of the arrayand the appropriate reagent is delivered to each of the reaction site.For example, the A amidite is delivered only to the sites where amiditeA is to be coupled during that synthesis step and so on. Common reagentsand washes are delivered by flooding the entire surface and thenremoving them by spinning

In still further embodiments, recognition moieties are spotted onto asuitable substrate. Such spotting can be done by hand with a capillarytube or micropipette, or by an automated spotting apparatus such asthose available from Affymetrix and Gilson (See e.g., U.S. Pat. Nos.5,601,980; 6,242,266; 6,040,193; and 5,700,637; each of which isincorporated herein by reference).

When the recognition moiety is an amine, in preferred embodiments, therecognition moiety will interact with a structure on the analyte whichreacts by binding to the amine (e.g., carbonyl groups, alkylhalogroups). In another preferred embodiment, the amine is protonated by anacidic moiety on the analyte of interest (e.g., carboxylic acid,sulfonic acid).

In certain preferred embodiments, when the recognition moiety is acarboxylic acid, the recognition moiety will interact with the analyteby complexation (e.g., metal ions). In still other preferredembodiments, the carboxylic acids will protonate a basic group on theanalyte (e.g. amine).

In another preferred embodiment, the recognition moiety is a drugmoiety. The drug moieties can be agents already accepted for clinicaluse or they can be drugs whose use is experimental, or whose activity ormechanism of action is under investigation. The drug moieties can have aproven action in a given disease state or can be only hypothesized toshow desirable action in a given disease state. In a preferredembodiment, the drug moieties are compounds that are being screened fortheir ability to interact with an analyte of choice. As such, drugmoieties that are useful in practicing the instant invention includedrugs from a broad range of drug classes having a variety ofpharmacological activities.

Classes of useful agents include, for example, non-steroidalanti-inflammatory drugs (NSAIDS). The MAIDS can, for example, beselected from the following categories: (e.g., propionic acidderivatives, acetic acid derivatives, fenamic acid derivatives,biphenylcarboxylic acid derivatives and oxicams); steroidalanti-inflammatory drugs including hydrocortisone and the like;antihistaminic drugs (e.g., chlorpheniranune, triprolidine); antitussivedrugs (e.g., dextromethorphan, codeine, carmiphen and carbetapentane);antipruritic drugs (e.g., methidilizine and trimeprizine);anticholinergic drugs (e.g., scopolamine, atropine, homatropine,levodopa); anti-emetic and antinauseant drugs (e.g., cyclizine,meclizine, chlorpromazine, buclizine); anorexic drugs (e.g.,benzphetamine, phentermine, chlorphentermine, fenfluramine); centralstimulant drugs (e.g., amphetamine, methamphetamine, dextroamphetamineand methylphenidate); antiarrhythmic drugs (e.g., propanolol,procainamide, disopyraminde, quinidine, encamide); P-adrenergic blockerdrugs (e.g., metoprolol, acebutolol, betaxolol, labetalol and timolol);cardiotonic drugs (e.g., milrinone, aminone and dobutamine);antihypertensive drugs (e.g., enalapril, clonidine, hydralazine,minoxidil, guanadrel, guanethidine); diuretic drugs (e.g., amiloride andhydrochlorothiazide); vasodilator drugs (e.g., diltazem, amiodarone,isosuprine, nylidrin, tolazoline and verapamil); vasoconstrictor drugs(e.g., dihydroergotamine, ergotamine and methylsergide); antiulcer drugs(e.g., ranitidine and cimetidine); anesthetic drugs (e.g., lidocaine,bupivacaine, chlorprocaine, dibucaine); antidepressant drugs (e.g.,imipramine, desipramine, amitryptiline, nortryptiline); tranquilizer andsedative drugs (e.g., chlordiazepoxide, benacytyzine, benzquinamide,flurazapam, hydroxyzine, loxapine and promazine); antipsychotic drugs(e.g., chlorprothixene, fluphenazine, haloperidol, molindone,thioridazine and trifluoperazine); antimicrobial drugs (antibacterial,antifungal, antiprotozoal and antiviral drugs).

Antimicrobial drugs which are preferred for incorporation into thepresent composition include, for example, pharmaceutically acceptablesalts of β-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin,tetracycline, erythromycin, amikacin, triclosan, doxycycline,capreomycin, chlorhexidine, chlortetracycline, oxytetracycline,clindamycin, ethambutol, hexamidine isothionate, metronidazole;pentamidine, gentamycin, kanamycin, lineomycin, methacycline,methenamine, minocycline, neomycin, netilmycin, paromomycin,streptomycin, tobramycin, miconazole, and amanfadine.

Other drug moieties of use in practicing the present invention includeantineoplastic drugs (e.g., antiandrogens (e.g., leuprolide orflutamide), cytocidal agents (e.g., adriamycin, doxorubicin, taxol,cyclophosphamide, busulfan, cisplatin, a-2-interferon) anti-estrogens(e.g., tamoxifen), antimetabolites (e.g., fluorouracil, methotrexate,mercaptopurine, thioguanine).

The recognition moiety can also comprise hormones (e.g.,medroxyprogesterone, estradiol, leuprolide, megestrol, octreotide orsomatostatin); muscle relaxant drugs (e.g., cinnamedrine,cyclobenzaprine, flavoxate, orphenadrine, papaverine, mebeverine,idaverine, ritodrine, dephenoxylate, dantrolene and azumolen);antispasmodic drugs; bone-active drugs (e.g., diphosphonate andphosphonoalkylphosphinate drug compounds); endocrine modulating drugs(e.g., contraceptives (e.g., ethinodiol, ethinyl estradiol,norethindrone, mestranol, desogestrel, medroxyprogesterone), modulatorsof diabetes (e.g., glyburide or chlorpropamide), anabolics, such astestolactone or stanozolol, androgens (e.g., methyltestosterone,testosterone or fluoxymesterone), antidiuretics (e.g., desmopressin) andcalcitonins).

Also of use in the present invention are estrogens (e.g.,diethylstilbesterol), glucocorticoids (e.g., triamcinolone,betamethasone, etc.) and progenstogens, such as norethindrone,ethynodiol, norethindrone, levonorgestrel; thyroid agents (e.g.,liothyronine or levothyroxine) or anti-thyroid agents (e.g.,methimazole); antihyperprolactinemic drugs (e.g., cabergoline); hormonesuppressors (e.g., danazol or goserelin), oxytocics (e.g.,methylergonovine or oxytocin) and prostaglandins, such as mioprostol,alprostadil or dinoprostone, can also be employed.

Other useful recognition moieties include immunomodulating drugs (e.g.,antihistamines, mast cell stabilizers, such as lodoxamide and/orcromolyn, steroids (e.g., triamcinolone, beclomethazone, cortisone,dexamethasone, prednisolone, methylprednisolone, beclomethasone, orclobetasol), histamine H₂ antagonists (e.g., famotidine, cimetidine,ranitidine), immunosuppressants (e.g., azathioprine, cyclosporin), etc.Groups with anti-inflammatory activity, such as sulindac, etodolac,ketoprofen and ketorolac, are also of use. Other drugs of use inconjunction with the present invention will be apparent to those ofskill in the art.

When the recognition moiety is a chelating agent, crown ether orcyclodextrin, host-guest chemistry will dominate the interaction betweenthe recognition moiety and the analyte. The use of host-guest chemistryallows a great degree of recognition-moiety-analyte specificity to beengineered into a device of the invention. The use of these compounds tobind to specific compounds is well known to those of skill in the art.See, for example, Pitt et al. “The Design of Chelating Agents for theTreatment of Iron Overload,” In, INORGANIC CHEMISTRY IN BIOLOGY ANDMEDICINE; Martell, A. E., Ed.; American Chemical Society, Washington,D.C., 1980, pp. 279-312; Lindoy, L. F., THE CHEMISTRY OF MACROCYCLICLIGAND COMPLEXES; Cambridge University Press, Cambridge, 1989; Dugas,H., BIOORGANIC CHEMISTRY; Springer-Verlag, New York, 1989, andreferences contained therein.

Additionally, a manifold of routes allowing the attachment of chelatingagents, crown ethers and cyclodextrins to other molecules is availableto those of skill in the art. See, for example, Meares et al.,“Properties of In Vivo Chelate-Tagged Proteins and Polypeptides.” In,MODIFICATION OF PROTEINS: FOOD, NUTRITIONAL, AND PHARMACOLOGICALASPECTS;” Feeney, R. E., Whitaker, l. R., Eds., American ChemicalSociety, Washington, D.C., 1982, pp. 370-38′7; Kasina et al.Bioconjugate Chem. 9:108-117 (1998); Song et al., Bioconjugate Chem.8:249-255 (1997).

In a presently preferred embodiment, the recognition moiety is apolyaminocarboxylate chelating agent such as ethylenediaminetetraaceticacid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). Theserecognition moieties can be attached to any amine-terminated componentof a SAM or a spacer arm, for example, by utilizing the commerciallyavailable dianhydride (Aldrich Chemical Co., Milwaukee, Wis.).

In still further preferred embodiments, the recognition moiety is abiomolecule such as a protein, nucleic acid, peptide or an antibody.Biomolecules useful in practicing the present invention can be derivedfrom any source. The biomolecules can be isolated from natural sourcesor can be produced by synthetic methods. Proteins can be naturalproteins or mutated proteins. Mutations can be effected by chemicalmutagenesis, site-directed mutagenesis or other means of inducingmutations known to those of skill in the art. Proteins useful inpracticing the instant invention include, for example, enzymes,antigens, antibodies and receptors. Antibodies can be either polyclonalor monoclonal. Peptides and nucleic acids can be isolated from naturalsources or can be wholly or partially synthetic in origin.

In those embodiments wherein the recognition moiety is a protein orantibody, the protein can be tethered to a SAM component or a spacer armby any reactive peptide residue available on the surface of the protein.In preferred embodiments, the reactive groups are amines orcarboxylates. In particularly preferred embodiments, the reactive groupsare the e-amine groups of lysine residues. Furthermore, these moleculescan be adsorbed onto the surface of the substrate or SAM by non-specificinteractions (e.g., chemisorption, physisorption).

Recognition moieties that are antibodies can be used to recognizeanalytes that are proteins, peptides, nucleic acids, saccharides orsmall molecules such as drugs, herbicides, pesticides, industrialchemicals and agents of war. Methods of raising antibodies for specificmolecules are well-known to those of skill in the art. See, U.S. Pat.Nos. 5,147,786; 5,334,528; 5,686,237; 5,573,922; each of which isincorporated herein by reference. Methods for attaching antibodies tosurfaces are also art-known (See, Delamarche et al. Langmuir12:1944-1946 (1996)).

Peptides and nucleic acids can be attached to a SAM component or spacerarm. Both naturally-derived and synthetic peptides and nucleic acids areof use in conjunction with the present invention. These molecules can beattached to a SAM component or spacer arm by any available reactivegroup. For example, peptides can be attached through an amine, carboxyl,sulfhydryl, or hydroxyl group. Such a group can reside at a peptideterminus or at a site internal to the peptide chain. Nucleic acids canbe attached through a reactive group on a base (e.g., exocyclic amine)or an available hydroxyl group on a sugar moiety (e.g., 3′- or5′-hydroxyl). The peptide and nucleic acid chains can be furtherderivatized at one or more sites to allow for the attachment ofappropriate reactive groups onto the chain (See, Chrisey et al. NucleicAcids Res. 24:3031-3039 (1996)).

When the peptide or nucleic acid is a fully or partially syntheticmolecule, a reactive group or masked reactive group can be incorporatedduring the process of the synthesis. Many derivatized monomersappropriate for reactive group incorporation in both peptides andnucleic acids are know to those of skill in the art (See, for example,THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY, Vol. 2: “Special Methods inPeptide Synthesis,” Gross, E. and Melenhofer, J., Eds., Academic Press,New York (1980)). Many useful monomers are commercially available(Bachem, Sigma, etc.). This masked group can then be unmasked followingthe synthesis, at which time it becomes available for reaction with aSAM component or a spacer arm. In other preferred embodiments, thepeptide is attached directly to the substrate

(See, Frey et al. Anal. Chem. 68:3187-3193 (1996)). In a particularlypreferred embodiment, the peptide is attached to a gold substratethrough a sulfhydryl group on a cysteine residue. In another preferredembodiment, the peptide is attached through a thiol to a spacer armwhich terminates in, for example, an iodoacetamide, chloroacetamide,benzyl iodide, benzyl bromide, alkyl iodide or alkyl bromide. Similarimmobilization techniques are known to those of skill in the art (See,for example, Zull et al. J. Ind Microbiol. 13:137-143 (1994)).

In another preferred embodiment, the recognition moiety forms aninclusion complex with the analyte of interest. In a particularlypreferred embodiment, the recognition moiety is a cyclodextrin ormodified cyclodextrin. Cyclodextrins are a group of cyclicoligosaccharides produced by numerous microorganisms. Cyclodextrins havea ring structure that has a basket-like shape. This shape allowscyclodextrins to include many kinds of molecules into their internalcavity (See, for example, Szejtli, J., CYCLODEXTRINS AND THEIR INCLUSIONCOMPLEXES; Akademiai Klado, Budapest, 1982; and Bender et al.,CYCLODEXTRIN CHEMISTRY, Springer-Verlag, Berlin, 1978).

Cyclodextrins are able to form inclusion complexes with an array oforganic molecules including, for example, drugs, pesticides, herbicidesand agents of war (See, Tenjarla et al., J. Pharm. Sci. 87:425-429(1998); Zughul et al., Pharm. Dev. Technol. 3:43-53 (1998); and Alberset al., Crit. Rev. Ther. Drug Carrier Syst. 12:311-337 (1995)).Importantly, cyclodextrins are able to discriminate between enantiomersof compounds in their inclusion complexes. Thus, in one preferredembodiment, the invention provides for the detection of a particularenantiomer in a mixture of enantiomers (See, Koppenhoefer et al. J.Chromatogr. A 793:153-164 (1998)).

The cyclodextrin recognition moiety can be attached to a SAM component,through a spacer arm or directly to the substrate (See, Yamamoto et al.,J. Phys. Chem. B 101:6855-6860 (1997)). Methods to attach cyclodextrinsto other molecules are well known to those of skill in thechromatographic and pharmaceutical arts (See, Sreenivasan, Appl. Polym.Sci. 60:2245-2249 (1996)).

IV. Mesogenic Layer

Any compound or mixture of compounds that forms a mesogenic layer can beused in conjunction with the present invention. The mesogens can formthermotropic or lyotropic liquid crystals. The mesogenic layer can beeither continuous or it can be patterned.

Both the thermotropic and lyotropic liquid crystals can exist in anumber of forms including nematic, chiral nematic, smectic, polarsmectic, chiral smectic, frustrated phases and discotic phases.

TABLE 1 Molecular structure of mesogens suitable for use in LiquidCrystal Assay Devices Mesogen Structure Anisaldazine

NCB

CBOOA

Comp A

Comp B

DB₇NO₂

DOBAMBC

nOm n = 1, m = 4: MBBA n = 2, m = 4: EBBA

nOBA n = 8: OOBA n = 9: NOBA

nmOBC

nOCB

nOSI

98P

PAA

PYP906

nSm

Presently preferred mesogens are displayed in Table 1. In a particularlypreferred embodiment, the mesogen is a member selected from the groupconsisting of 4-cyano-4′-pentylbiphenyl,N-(4-methoxybenzylidene)-4-butlyaniline and combinations thereof.

The mesogenic layer can be a substantially pure compound, or it cancontain other compounds that enhance or alter characteristics of themesogen. Thus, in one preferred embodiment, the mesogenic layer furthercomprises a second compound, for example and alkane, which expands thetemperature range over which the nematic and isotropic phases exist. Useof devices having mesogenic layers of this composition allows fordetection of the analyte recognition moiety interaction over a greatertemperature range.

In some preferred embodiments, the mesogenic layer further comprises adichroic dye or fluorescent compound. Examples of dichroic dyes andfluorescent compounds useful in the present invention include, but arenot limited to, azobenzene, BTBP, polyazocompounds, anthraquinone,perylene dyes, and the like. In particularly preferred embodiments, adichroic dye of fluorescent compound is selected that complements theorientation dependence of the liquid crystal so that polarized light isnot required to read the assay. In some preferred embodiments, if theabsorbance of the liquid crystal is in the visible range, then changesin orientation can be observed using ambient light without crossedpolars. In other preferred embodiments, the dichroic dye or fluorescentcompound is used in combination with a fluorimeter and the changes influorescence are used to detect changes in orientation of the liquidcrystal.

In another preferred embodiment, the analyte first interacts with therecognition moiety and the mesogenic layer is introduced in itsisotropic phase. The mesogenic layer is subsequently cooled to form theliquid crystalline phase. The presence of the analyte within regions ofthe mesogenic layer will disturb the equilibrium between the nematic andisotropic phases leading to different rates and magnitudes of nucleationat those sites. The differences between the nematic and isotropicregions are clearly detectable.

V. Patterned Liquid Crystals

One approach to the patterning of the mesogenic layer on flat and curvedsurfaces is based on the use of patterned SAMs of molecules to directboth the polar (away from the surface) and azimuthal (in the plane ofthe surface) orientations of the mesogenic layer. This method is simpleand flexible, and any of the recently established procedures forpatterning SAMs on surfaces (for example, microcontact printing orphoto-patterning) (Talov et al., J. Am. Chem. Soc. 115: 5305 (1993);Kumar et al., Acc. Chem. Res. 28: 219 (1995), and references therein;Xia et al., J. Am. Chem. Soc. 117: 3274 (1995), and references thereincan be used; Jackman et al., Science 269: 664 (1995)). Using any ofthese methods, SAMs which pattern liquid crystals can be easily extendedto sizes ranging from hundreds of nanometers (Xia et al., J. Am. Chem.Soc. 117: 3274 (1995), and references therein) to millimeters and permitboth planar (parallel to the surface) and homeotropic (perpendicular tothe surface) orientation of mesogenic layers; methods based on therubbing of polymer films mainly provide manipulation of the in-planealignment of mesogenic layers and cannot homeotropically align mesogeniclayers. One class of useful SAMs has surface energies (˜19 mJ/m²) abouthalf those of films of polyimides used for alignment of liquid crystals;low-energy surfaces are less prone to contamination by molecularadsorbates and dust particles than are high-energy ones. Because SAMscan also be patterned on non-planar surfaces (Jackman et al., Science269: 664 (1995)), patterned mesogenic structures formed with SAMs can bereplicated on curved surfaces.

The capacity to pattern mesogenic layer orientations on nonplanarsurfaces provides procedures for the fabrication of tunable hybriddiffractive-refractive devices. For example, devices based oncombinations of diffractive and refractive optical processes permitaplanatic or chromatic correction in lenses, spectral dispersion,imaging from a single optical element, and other manipulations of light(Resler et al., Opt. Lett. 21, 689 (1996); S. M. Ebstein, ibid., p.1454; M. B. Stem, Microelectron. Eng. 32, 369 (1996): Goto et al., Jpn.J. Appl. Phys. 31, 1586 (1992); Magiera et al., Soc. Photo-Opt. Instrum.Eng., 2774, 204 (1996)). The capability to pattern mesogenic layers oncurved surfaces also provides routes for the fabrication of displayswith wide viewing angles.

In a presently preferred embodiment, the tunable hybrid device permitsthe manipulation of light. In a further preferred embodiment, the deviceis a refractive-diffractive device. In a still further preferredembodiment, the device permits imaging from a single optical element. Inyet another preferred embodiment, the device permits aplanatic orchromatic correction in lenses. In still another preferred embodiment,the device allows for spectral dispersion.

In another presently preferred embodiment, the SAM is layered on amaterial suitable for use as an electrode. In a preferred embodiment,the material is a metal film. In a further preferred embodiment, themetal film is a gold film.

The patterned mesogenic layers of the instant invention can be tuned bythe use of electric fields. In a preferred embodiment, the electricfield is used to reversibly orient the mesogenic layer. In a stillfurther preferred embodiment, the electric field is applied eitherperpendicular to, or in the plane of, the surface of the mesogeniclayer. In another preferred embodiment, the oriented mesogenic layermodulates the intensity of light diffracted from the layer.

The discussion above, concerning SAM components, SAM components withreactive groups and SAM components bearing recognition moieties isequally applicable in the context of this aspect of the invention. Thus,the constituents of the SAM can be chosen from any of a wide variety ofappropriate molecules. In a presently preferred embodiment, the SAMcomprises mixtures of R²¹CH₂(CH₂)₁₄SH and R³¹CH₂(CH₂)₁₅SH, where R²¹ andR³¹ are independently members elected from the group consisting ofhydrogen, reactive groups and recognition groups, as discussed above.

VI. Analytes

It is contemplated that the devices and methods of the present inventioncan be used to detect any analyte, or class of analytes, which interactwith a recognition moiety in a manner that perturbs the mesogenic layerin a detectable manner. This statement does not appear to includenon-specific interactions. The interaction between the analyte andrecognition moiety can be any physicochemical interaction, includingcovalent bonding, ionic bonding, hydrogen bonding, van der Waalsinteractions, repulsive electronic interactions, attractive electronicinteractions and hydrophobic/hydrophilic interactions.

In a preferred embodiment, the interaction is an ionic interaction. Inthis embodiment, an acid, base, metal ion or metal ion-binding ligand isthe analyte. In a still further preferred embodiment, the interaction isa hydrogen bonding interaction. In a particularly preferred embodiment,the hybridization of an immobilized nucleic acid to a nucleic acidhaving a complementary sequence is detected. In another preferredembodiment, the interaction is between an enzyme or receptor and a smallmolecule that binds thereto.

In another embodiment, the analyte competes for the recognition moietywith another agent, which has been bound to the recognition moiety priorto introducing the analyte of interest. In this embodiment, it is theprocess or result of the analyte displacing the pre-bound agent, whichcauses the detectable perturbation in the mesogenic layer. Suitablecombinations of recognition moieties and analytes will be apparent tothose of skill in the art.

In presently preferred embodiments, the analyte is a member selectedfrom the group consisting of acids, bases, organic ions, inorganic ions,pharmaceuticals, herbicides, pesticides, chemical warfare agents,noxious gases, biomolecules (e.g., polypeptides, carbohydrates, andpolynucleotides) and microorganisms (e.g., viruses, bacteria, prions,mycoplasmas, etc.). Importantly, each of these agents can be detected asa vapor or in a liquid solution. These agents can be present ascomponents in mixtures of structurally unrelated compounds, racemicmixtures of stereoisomers, non-racemic mixtures of stereoisomers,mixtures of diastereomers, mixtures of positional isomers or as purecompounds. Within the scope of the invention is a device and a method todetect a particular analyte of interest without interference from othersubstances within a mixture.

Both organic and inorganic acids can be detected using the device andmethod of the present invention. In a preferred embodiment, therecognition moiety comprises a group that is protonated by the acid. Theresult of this protonation is a detectable perturbation in theconfiguration of the mesogenic layer. While not wishing to be bound byany particular theory of operation, the inventors currently believe thatthis perturbation can be achieved by a change in the size orconformation of the recognition moiety on protonation. Alternatively,the protonation may induce repulsion between proximate recognitionmoieties bearing charges of the same sign. Further, the protonation caninduce an overall positive charge across the SAM, which perturbs theelectronic distribution of the molecules in the mesogenic layer. Thisperturbation can be due to an electronic redistribution in the mesogenicmolecules or can be due to repulsive or attractive interaction between acharged mesogen and a similarly, or oppositely, charged SAM.

In another preferred embodiment, the invention provides a device and amethod for detecting bases. The methods for the detection and themechanisms which allow such detection of bases are substantially similarto those discussed above in the context of acid detection; the notableexception being that the base will preferably deprotonate a group on aSAM component, spacer arm or substrate.

Organic ions that are substantially non-acidic and non-basic (e.g.,quaternary alkylammonium salts) can be detected by a recognition moiety.For example, a recognition moiety with ion exchange properties is usefulin the present invention. A specific example is the exchange of a cationsuch as dodecyltrimethylammonium cation for a metal ion such as sodium,using a SAM presenting. Recognition moieties that form inclusioncomplexes with organic cations are also of use. For example, crownethers and cryptands can be used to form inclusion complexes withorganic ions such as quaternary ammonium canons.

Inorganic ions such as metal ions and complex ions (e.g., SO₄, PO₄) canalso be detected using the device and method of the invention. Metalions can be detected, for example, by their complexation or chelanon byagents bound to a SAM component, spacer arm or the substrate. In thisembodiment, the recognition moiety can be a simple monovalent moiety(e.g., carboxylate, amine, thiol) or can be a more structurally complexagent (e.g., ethylenediaminepentaacetic acid, crown ethers, aza crowns,thia crowns). The methods of detection and the mechanisms allowing suchdetection are substantially similar to those discussed in the context ofacid detection.

Complex inorganic ions can be detected by their ability to compete withligands for bound metal ions in ligand-metal complexes. When a ligandbound to a SAM component, a spacer arm or a substrate forms ametal-complex having a thermodynamic stability constant which is lessthan that of the complex between the metal and the complex ion, thecomplex ion will cause the dissociation of the metal ion from theimmobilized ligand. The dissociation of the metal ion will perturb themesogenic layer in a detectable manner. Methods of determining stabilityconstants for compounds formed between metal ions and ligands are wellknown to those of skill in the art. Using these stability constants,devices that are specific for particular ions can be manufactured. See,Martell, A. E., Motekaitis, R. J., DETERMINATION AND USE OF STABILITYCONSTANTS, 2d Ed., VCH Publishers, New York 1992.

Small molecules such as pesticides, herbicides, agents of war, and thelike can be detected by the use of a number of different recognitionmoiety motifs. Acidic or basic components can be detected as describedabove. An agent's metal binding capability can also be used toadvantage, as described above for complex ions. Additionally, if theseagents bind to an identified biological structure (e.g., a receptor),the receptor can be immobilized on the substrate, a SAM component or aspacer arm. Techniques are also available in the art for raisingantibodies that are highly specific for a particular small molecule.Thus, it is within the scope of the present invention to make use ofantibodies against small molecules for detection of those molecules.

In a preferred embodiment, the affinity of an analyte for a particularmetal ion is exploited by having a SAM component, spacer arm orsubstrate labeled with an immobilized metal ion. The metal ion generallymust have available at least one empty coordination site to which theanalyte can bind. Alternatively, at least one bond between the metal andthe metal-immobilizing agent must be sufficiently labile in the presenceof the analyte to allow the displacement of at least one bond of theimmobilizing reagent by the analyte.

In a preferred embodiment, the agent detected by binding to animmobilized metal ion is an organophosphorous compound such as aninsecticide or an agent of war (e.g., VX,O-ethyl-S-(2-diisopropylaminoethyl)methylthiophosphonate). Exemplarycompounds which exhibit affinity for organophosphorous agents include,but are not limited to, Cu⁺²-diamine,triethylentetraamine-Cu⁺²-chloride, tetraethylenediamine-Cu⁺²-chlorideand 2,2′ bipyridine-Cu⁺²-chloride (U.S. Pat. No. 4,549,427, incorporatedherein by reference).

In another preferred embodiment, antibodies to the particular agents areimmobilized on the substrate, a SAM component or a spacer arm.Techniques for raising antibodies to herbicides, pesticides and agentsof war are known to those of skill in the art. See, Harlow, Lane,MONOCLONAL ANTIBODIES: A LABORATORY MANUAL, Cold Springs HarborLaboratory, Long Island, N.Y., 1988.

In a preferred embodiment, the herbicides are preferably members of thegroup consisting of triazines, haloacetanilides, carbamates, toluidines,areas, plant growth hormones and diphenyl ethers. Included within thesebroad generic groupings are commercially available herbicides such asphenoxyl alkanoic acids,

bipyridiniums, benzonitriles, dinitroanilines, acid amides, carbamates,thiocarbamates, heterocyclic nitrogen compounds including triazines,pyridines, pyridazinones, sulfonylureas, imidazoles, substituted areas,halogenated aliphatic carboxylic acids, inorganics, organometallics andderivatives of biologically important amino acids.

In the embodiments discussed above, the preferred agent of war is amember of the group consisting of mustard and related vesicantsincluding the agents known as HD, Q, T, HN1, HN2, HN3, nerve agents,particularly the organic esters of substituted phosphoric acid includingtabun, sarin, isopropyl methylphosphonofluoridate, soman pinacolylmethylphosphonofluoridate. Other detectable analytes includeincapacitants such as BZ, 3-quinuclidinyl benzilate and irritants suchas the riot control compound CS.

Pesticides preferred for detection using the present invention includebactericides (e.g., formaldehyde), fumigants (e.g., bromomethane),fungicides (e.g., 2-phenylphenol, biphenyl, mercuric oxide, imazalil),acaricides (e.g., abamectin, bifenthrin), insecticides (e.g.,imidacloprid, prallethrin, cyphenothrin)

The present invention also provides a device and a method for detectingnoxious gases such as CO, CO₂, SO₃, H₂SO₄, SO₂, NO, NO₂, N₂O₄ and thelike. In a preferred embodiment, the SAM, the substrate or a spacer armincludes at least one compound capable of detecting the gas. Usefulcompounds include, but are not limited to, palladium compounds selectedfrom the group consisting of palladium sulfate, palladium sulfite,palladium pyrosulfite, palladium chloride, palladium bromide, palladiumiodide, palladium perchlorate, palladium complexes with organiccomplexing reagents and mixtures thereof.

Other compounds of use in practicing this embodiment of the presentinvention include, molybdenum compounds such as silicomolybdic acid,salts of silicomolybdic acid, molybdenum trioxide, heteropolyacids ofmolybdenum containing vanadium, copper or tungsten, ammonium molybdate,alkali metal or alkaline earth salts of molybdate anion,heteropolymolybdates and mixtures thereof.

Still further useful gas detecting compounds include, copper salts andcopper complexes with an available coordination site.Alpha-cyclodextrin, betacyclodextrin, modified alpha- andbeta-cyclodextrins, gamma-cyclodextrin and mixtures thereof are of usein practicing the present invention (U.S. Pat. Nos. 5,618,493, and5,071,526, each of which is incorporated herein by reference).

In another preferred gas detecting embodiment, the substrate, SAMcomponent or spacer arm is derivatized with a compound selected from thegroup consisting of amorphous hemoglobin, crystalline hemoglobin,amorphous heme, crystalline heme and mixtures thereof. The heme servesas a recognition moiety that is reactive towards the gas (U.S. Pat. No.3,693,327, incorporated herein by reference).

When the analyte is a biomolecule, any recognition moiety that interactswith the biomolecule is useful in practicing the present invention.Thus, when the analyte is a nucleic acid, in one embodiment, therecognition moiety is a nucleic acid having a sequence that is at leastpartially complementary to the recognition moiety sequence. When therecognition moiety is a peptide, an antibody specific for that peptidecan be used as the analyte. In another preferred embodiment, a protein,other than an antibody (e.g., enzyme, receptor) is the analyte.

In a presently preferred embodiment, the recognition moiety interactswith biotin and is avidin or an anti-biotin antibody. Other recognitionmoieties of use when the analyte is a biomolecule will be apparent tothose of skill in the art.

In still further preferred embodiments, microorganisms, includingpathogens are detected. In some embodiments, the recognition moiety usedto detect microorganisms is an antibody directed to the microorganism.In other embodiments, ligands are incorporated to detect a variety ofpathogenic organisms including, but not limited to, sialic acid todetect HIV (Wies et al., Nature 333: 426 [1988]), influenza (White etal., Cell 56: 725 [1989]), Chlamydia (Infect. Imm. 57: 2378 [1989]),Neisseria meningitidis, Streptococcus suis, Salmonella, mumps,newcastle, and various viruses, including reovirus, Sendai virus, andmyxovirus; and 9-OAC sialic acid to detect coronavirus,encephalomyelitis virus, and rotavirus; non-sialic acid glycoproteins todetect cytomegalovirus (Virology 176: 337 [1990]) and measles virus(Virology 172: 386 [1989]); CD4 (Khatzman et al., Nature 312: 763[1985]), vasoactive intestinal peptide (Sacerdote et al., J. ofNeuroscience Research 18: 102 [1987]), and peptide T (Ruff et al., FEBSLetters 211: 17 [1987]) to detect HIV; epidermal growth factor to detectvaccinia (Epstein et al., Nature 318: 663 [1985]); acetylcholinereceptor to detect rabies (Lentz et al., Science 215: 182 [1982]); Cd3complement receptor to detect Epstein-Barr virus (Carel et al., J. Biol.Chem. 265: 12293 [1990]); -adrenergic receptor to detect reovirus (Co etal., Proc. Natl. Acad. Sci. 82: 1494 [1985]); ICAM-1 (Marlin et al.,Nature 344: 70 [1990]), N-CAM, and myelin-associated glycoprotein MAb(Shephey et al., Proc. Natl. Acad. Sci. 85: 7743 [1988]) to detectrhinovirus; polio virus receptor to detect polio virus (Mendelsohn etal., Cell 56: 855 [1989]); fibroblast growth factor receptor to detectherpes virus (Kaner et al., Science 248: 1410 [1990]); oligomannose todetect Escherichia coli; ganglioside G to detect Neisseria meningitidis;and antibodies to detect a broad variety of pathogens (e.g., Neisseriagonorrhoeae, V. vulnificus, V. parahaemolyticus, V. cholerae, and V.alginolyticus).

VII. Compound Libraries

The synthesis and screening of chemical libraries to identify compoundsthat have novel pharmacological and material science properties is acommon practice. Libraries that have been synthesized include, forexample, collections of oligonucleotides, oligopeptides, and small orlarge molecular weight organic or inorganic molecules. See WO 97/35198,WO 96/40732, and Gallop et al., J. Med. Chem. 37:1233-51 (1994).

Thus, in some embodiments, the invention provides a device forsynthesizing and screening a library of compounds, comprising:

(1) a synthesis component, comprising:

(a) a first substrate having a surface;

(b) a self-assembled monolayer on the surface, said monolayer comprisinga reactive functionality; and

(2) an analysis component, comprising:

(a) a second substrate having a surface; and

(b) a mesogenic layer between said surface of said first substrate andsaid surface of said second substrate.

In a preferred embodiment, the second substrate has a self-assembledmonolayer attached thereto. In yet another preferred embodiment, thesecond substrate is permeable to liquids, vapors, gases and combinationsthereof. The permeable substrate allows analytes to come into contactwith the self-assembled monolayer(s) and the mesogenic layer, whilemaintaining the overall integrity of the optical cell.

The discussion above concerning substrates, organic layers and mesogeniclayers is applicable to each of the embodiments of this aspect of theinvention. In a presently preferred embodiment, the substrate comprisesa metal film. In a further preferred embodiment, the metal film is amember selected from the group consisting of gold, nickel, platinum,silver, palladium and copper. In a still further preferred embodiment,the metal film is obliquely deposited.

The organic layer can be constructed of any organic substance whichassociates with the substrate, preferably, the organic layerconstituents are moieties selected from the group consisting ofalkanethiols, functionalized alkanethiols and combinations thereof. In afurther preferred embodiment, at least one component of the organiclayer is a moiety which is a member selected from the group consistingof R²¹CH₂(CH₂)₁₄SH and R³¹CH₂(CH₂)₁₅SH, wherein R²¹ and R³¹ areindependently members selected from the group consisting of hydrogen,reactive groups and recognition moieties.

The discussion above concerning reactive groups is equally applicable tothis aspect of the invention. In certain preferred embodiments, R²¹ andR³¹ are independently members selected from the group consisting ofhydrogen, amine, carboxylic acid, carboxylic acid derivatives, alcohols,thiols, alkenes and combinations thereof.

The SAM can be patterned by any of the above-discussed methods forproducing patterned substrates and organic layers. The discussion aboveconcerning the patterning of substrates and the construction of organiclayers from a mixture of components having different properties isgenerally applicable to this embodiment of the invention. In a presentlypreferred embodiment, the SAM is patterned by microcontact printing. Ina further preferred embodiment, the microcontact printing utilizes acomponent that is distinct from the components of the self-assembledmonolayer.

The mesogenic layer can comprise one or more mesogenic compounds. Thediscussion above concerning the nature of the mesogenic layer isgenerally applicable to this embodiment of the invention. In a presentlypreferred embodiment, the mesogenic layer comprises a mesogen which is amember selected from the group consisting of 4-cyano-4′-pentylbiphenyl,N-(4-methoxybenzylidene)-4-butylanailine and combinations thereof.

In another preferred embodiment, the present invention provides a methodfor synthesizing and analyzing a combinatorial library of compoundsusing the above described device. The method comprises,

(a) adding a first component of a first compound to a first region ofsaid surface of said first substrate and a first component of a secondcompound to a second region of said surface of said first substrate;

(b) adding a second component of said first compound to said firstregion of said surface of said first substrate and adding a secondcomponent of said second compound to said second region on said surfaceof said first substrate;

(c) reacting said first and second components to form a first productand a second product;

(d) applying said mesogenic layer to said surface of said firstsubstrate;

(e) adding an analyte to said first region and said second region; and

(f) detecting said switch in said mesogenic layer from a firstorientation to said second orientation, whereby said analyzing isachieved.

The sequential addition of components can be repeated as many times asnecessary in order to assemble the desired library of compounds.Additionally, any number of solvents, catalysts and reagents necessaryto effect the desired molecular transformations can be added before,concurrently or after the addition of the component.

Virtually any type of compound library can be synthesized using themethod of the invention, including peptides, nucleic acids, saccharides,and small and large molecular weight organic and inorganic compounds.

In a presently preferred embodiment, when the synthesis is complete, asecond substrate is layered on top of the mesogenic layer. In a furtherpreferred embodiment, the second substrate has an attached secondself-assembled monolayer that contacts the mesogenic layer. Thediscussion above concerning the permutations available when twosubstrates are utilized is generally applicable to this embodiment. In astill further preferred embodiment, the second substrate is a permeablesubstrate. In yet another preferred embodiment, the second substrate ispatterned similar to the first substrate.

In a presently preferred embodiment, the libraries synthesized comprisemore than 10 unique compounds, preferably more than 100 unique compoundsand more preferably more than 1000 unique compounds.

In still further embodiments, the present invention also provides alibrary of compounds synthesized on a self-assembled monolayer. Thediscussion above concerning libraries, SAMs, functionalized SAMcomponents, mesogenic layers, and the like is generally applicable tothis aspect of the invention.

VIII. The Device

The device of the present invention can be of any configuration thatallows for the contact of a mesogenic layer with an organic layer orinorganic layer (e.g., metal, metal salt or metal oxide). The onlylimitations on size and shape are those that arise from the situation inwhich the device is used or the purpose for which it is intended. Thedevice can be planar or non-planar. Thus, it is within the scope of thepresent invention to use any number of polarizers, lenses, filterslights, and the like to practice the present invention.

Although many changes in the mesogenic layer can be detected by visualobservation under ambient light, any means for detecting the change inthe mesogenic layer can be incorporated into, or used in conjunctionwith, the device. Thus, it is within the scope of the present inventionto use lights, microscopes, spectrometry, electrical techniques and thelike to aid in the detection of a change in the mesogenic layer.

In those embodiments utilizing light in the visible region of thespectrum, the light can be used to simply illuminate details of themesogenic layer. Alternatively, the light can be passed through themesogenic layer and the amount of light transmitted, absorbed orreflected can be measured. The device can utilize a backlighting devicesuch as that described in U.S. Pat. No. 5,739,879, incorporated hereinby reference. Light in the ultraviolet and infrared regions is also ofuse in the present invention.

Thus, in another aspect, the invention provides a method for varying theoptical texture of a mesogenic layer comprising a haloorganosulfur. Thehaloorganosulfur has a halogen content. The optical texture of themesogenic layer is controlled by selecting the halogen content of thehaloorganosulfur.

The present invention contemplates the use of plate readers to detectchanges in the orientation of mesogens upon binding of an analyte. Theplate readers may be used in conjunction with the LC assay devicesdescribed herein and also with the lyotropic LC assays described in U.S.Pat. No. 6,171,802, incorporated herein by reference. In particular, thepresent invention includes methods and processes for the quantificationof light transmission through films of liquid crystals based onquantification of transmitted or reflected light.

The present invention is not limited to any particular mechanism ofaction. Indeed, an understanding of the mechanism of action is notrequired to practice the present invention. Nevertheless, it iscontemplated that ordered nanostructured substrates impart order to thinfilms of liquid crystal placed onto their surface. These ordered filmsof liquid crystal preserve the plane of polarized light passed throughthem. If the liquid crystal possesses a well-defined distortion—such asa 90 degree twist distortion—then the liquid crystal will change thepolarization of the transmitted light in a well-defined and predictablemanner. It is further contemplated that ordered films of liquid crystaldifferentially absorb (relative to randomly ordered films of liquidcrystal) specific wavelengths of light.

In some embodiments of the present invention, the amount of targetmolecule or molecules bound to a sensing surface of an LC assay device(i.e., a surface decorated with a recognition moiety) increases with theconcentration/amount of target molecule present in a sample in contactwith a sensing surface. In preferred embodiments, the amount of boundtarget molecule changes the degree of disorder introduced into a thinfilm of liquid crystal that is ordered by nature of the underlyingnanostructured sensing substrate. In some embodiments, the degree oforder present in a thin film of liquid crystal determines the amount oflight transmitted through the film when viewed through crossed polars.In other embodiments, the degree of order present in a thin film ofliquid crystal determines the amount of light transmitted through thefilm when viewed using specific wavelengths of light. In still otherembodiments, the reflectivity of an interface to a liquid crystal canchange with the orientation of the liquid crystal. Therefore, in someembodiments, oblique illumination of the LC assay device is utilizedwith collection and analysis of reflected light being performed.

Accordingly, the present invention contemplates the use of plate readersto detect light transmission through an LC assay device when viewedthrough cross polars, the transmission of light through an LC assaydevice illuminated with a suitable wavelength of light, or reflection oflight (i.e., polarized light or non-polarized light of specificwavelengths) from the surface of an LC assay device. In particularlypreferred embodiments, plate readers are provided that are designed tobe used in conjunction with LC assays. Other embodiments of the presentinvention provide modified commercially available readers such as ELISAreaders and fluorometric readers adapted to read LC assays.

Non-limiting examples of the plate readers of the present invention areprovided in FIGS. 1 and 2. In preferred embodiments, two polarizingfilters are placed in the optical pathway of the plate reader in acrossed or parallel polar configuration. One filter is placed on theemission side of the light path prior to passing through the samplewhile a second polarizing filter is placed on the analyzing side of thelight path after light has passed through the sample but before it iscollected by a sensing devise such as a photodiode or a CCD. An orderedliquid crystal in the LC assay device preserves the plane ofpolarization and the amount of light reaching the light gathering andsensing device is markedly attenuated when viewed through cross polarsor markedly accentuated when viewed through parallel polars. Randomorganization of the liquid crystal of the LC assay device does notpreserve the plane of polarization and the amount of light, passingthrough crossed polars, reaching the light collecting and sensing deviceis relatively unaffected. Accordingly, in preferred embodiments, thebinding of target molecules by the recognition moieties in an LC assaydevice introduces disorder into the overlying thin film of LC thatincreases with the amount of bound target molecule. In otherembodiments, specific bandpass filters are placed on the excitation sideof the light path before light encounters the sample as well as on theemission side of the light path (after light has passed through or isreflected by the sample but before reaching the light collecting andsensing device (e.g., photodiode or CCD). This configuration is usefulfor quantifying both reflected and transmitted light

The present invention also provides LC assay devices configured for usein the plate reader. In preferred embodiments, the LC assay device isformatted or arrayed according to the dimensions of standardcommercially available plates (e.g., 24, 96, 384 and 1536 well plates).In some embodiments, the LC assay device comprises a surface (e.g., asubstrate with recognition moieties attached) that is of proper externaldimensions to be accurately fit into a given commercial reader. In someembodiments, the substrate contains uniform topography across itssurface, while in other embodiments, the substrate contains a gradientof topographies across its surface. The recognition moieties may bearrayed on the substrate surface in any appropriate configuration. Forexample, in some embodiments, a single binding antibody, polypeptide, orpolynucleotide is evenly distributed across the surface. In otherembodiments, a single binding antibody, polypeptide, or polynucleotideis distributed across the surface in a gradient. In still otherembodiments, a single binding antibody, polypeptide, or polynucleotideis arrayed in discrete spots that are in proper alignment to be read bythe commercial reader. In still further embodiments, a variety ofdifferent antibodies, polypeptides, or polynucleotides are arrayed inspots that are in proper alignment to read by the commercial reader. Instill other embodiments, a variety of different antibodies,polypeptides, or polynucleotides are arrayed in zones along the surface.In the drawing below each zone would contain a different antibody orbinding sequence. The plate would be read at predetermined points (e.g.,spots corresponding to the location of the wells in a 96 well plate). Bydesigning the zones to the configuration of the plate reader it will beknown which “well” readings correspond to each zone. In otherembodiments, specifically designed well inserts (to be used withcommercially available 24, 96, 384 or 1536 well plates) containing thenanostructured sensing surface will be used in conjunction withcommercially available multiwell plates for performing the LC assays.

It will also be recognized that the present invention provides an assaysystem comprising a plate reading device and an LC assay device, whereinthe plate reading device and LC assay device are configured so thatlight provided from the plate reading device which is passed through orreflected from at least one surface of the LC assay device is detectedby a detection unit of the plate reading device. Suitable detectingunits include CCDs and photomultiplier tubes.

Commercially available plate readers that may be modified according tothe present invention include, but are not limited, to those availablefrom Nalge Nunc International Corporation (Rochester, N.Y.), GreinerAmerica, Inc. (Lake Mary, Fla.), Akers Laboratories Inc., (Thorofare,N.J.), Alpha Diagnostic International, Inc. (San Antonio, Tex.), andQiagen Inc. (Valencia, Calif.).

X. Quantitation of Analytes

The present invention provides devices and methods for quantitating theamount an analyte in a sample. In some embodiments, the devices of thepresent invention include electrodes for applying an electrical fieldacross the liquid crystal. It is contemplated that the thresholdelectrical field (applied voltage) required to detect (e.g., opticallyor electrically) the onset of reorientation of the liquid crystal willbe correlated to the presence of bound analyte. Without being bound toany particular theory, it is believed the presence of the bound analytewill change the strength of anchoring of the liquid crystal andtherefore be useful in both detecting a bound analyte, and inparticularly preferred embodiments, quantifying the amount of boundanalyte.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); C(degrees Centigrade); U (units), mU (milliunits); min. (minutes); sec.(seconds); % (percent); kb (kilobase); by (base pair); PCR (polymerasechain reaction); BSA (bovine serum albumin).

Example 1

This Example describes the detection of non-specific adsorption of amolecule to a surface. An electron beam evaporator is used to obliquelydeposit 30 Angstroms of titanium and then 130 Angstroms of gold ontoimmobile glass microscope slides. The rate of deposition of the metalsis 0.2 Angstroms/sec and the angle of deposition is 50 degrees from thenormal. This gold film is immersed into an ethanolic solution ofhexadecanethiol to form a hydrophobic monolayer on the surface of thegold film. A droplet of 10 micromolar BSA in PBS is then placed on thesurface of the hydrophobic gold film for 20 mins. The droplet is thenrinsed from the surface using PBS, then water. The functionalized goldfilm is then paired with a second gold film that supports aself-assembled monolayer formed from decanethiol and hexadecanethiolthat causes nematic phases of 5CB to orient perpendicular to the mixedmonolayer. A thin film of Mylar (thickness 10-20 micrometers) is used toseparate the two surfaces and secured using a bulldog clip. 5CB isheated into its isotropic phase and introduced into the cavity betweenthe two surfaces by using capillary forces, and then the 5CB is allowedto cool to room temperature, thus forming a nematic phase. The liquidcrystal cell is placed into a polarized light microscope and viewedbetween crossed-polars (in transmission). The area of the hydrophobicmonolayer on which the droplet of BSA causes the liquid crystal toassume a non-uniform orientation whereas the region of the surface thatdoes not possess BSA non-specifically adsorbed to the surface causes auniform alignment of the liquid crystal. Thus, the liquid crystal isdemonstrated to be useful for imaging of non-specific adsorption of BSAto the surface.

Example 2

This Example describes the detection of non-specific adsorption of amolecule to a surface. In particular, this example describes detectionof the non-specific binding of the BSA to the SAM formed fromhexadecanethiol. An electron beam evaporator is used to obliquelydeposit 30 Angstroms of titanium and then 130 Angstroms of gold ontoimmobile glass microscope slides. The rate of deposition of the metalsis 0.2 Angstroms/sec and the angle of deposition is 50 degrees from thenormal. This gold film is immersed into an ethanolic solution ofhexadecanethiol to form a hydrophobic monolayer on the surface of thegold film. Next, BSA is adsorbed to the gold film at a level that doesnot cause liquid crystal on the surface to assume a non-uniformorientation. Anti-BSA IgG in Triton X-100 (to prevent non-specificadsorption of the IgG) is then bound to the BSA. The bound anti-biotinIgG is imaged by placing liquid crystal on the surface.

Example 3

This Example describes the detection of non-specific adsorption of amolecule to a surface. First, a cellulose nitrate film is mechanicallyrubbed so that it uniformly aligns liquid crystal. Proteins separated bygel electrophoresis are transferred onto the surface of the rubbed filmby standard Western blotting procedures. The bands of transferredproteins are imaged by placement of liquid crystal on the rubbed film.

Example 4

This Example describes the detection of non-specific adsorption of amolecule to a surface. An electron beam evaporator is used to obliquelydeposit 30 Angstroms of titanium and then 130 Angstroms of gold ontoimmobile glass microscope slides. The rate of deposition of the metalsis 0.2 Angstroms/sec and the angle of deposition is 50 degrees from thenormal. Monolayers formed from HS(CH₂)₈N⁺(CH₃)₃ and HS(CH₂)₂SO₃ ⁻ arethen patterned on the surface to form regions that present SO₃ ⁻ orN⁺(CH₃)₃ groups. A microfluidic channel molded in PDMS is then placed onthe patterned surface. Two proteins are flowed across the patternedsurface at a pH such that one protein is above its pI whereas the otherprotein is below it. Thus one protein will adsorb onto the negativelycharged region of the surface whereas the other protein will adsorb ontothe positively charged region. An example of a protein pair iscytochrome-C (pI=10.7) and beta-lactoglobulin (pI=5.2), and use of PBSat 6.9. The surface is rinsed with PBS and then with water. Binding isimaged with a liquid crystal by forming a liquid crystal cell.

Example 5

This Example describes the detection of non-specific adsorption of amolecule to a surface. An electron beam evaporator is used to obliquelydeposit 30 Angstroms of titanium and then 130 Angstroms of gold ontoimmobile glass microscope slides. The rate of deposition of the metalsis 0.2 Angstroms/sec and the angle of deposition is 50 degrees from thenormal. A region of biotin-terminated monolayers is patterned on thesurface of the gold. The remainder of the surface is filled in usinghexadecanethiol (so as to create a hydrophobic surface). Next, using amicrofluidic channel molded in PDMS, anti-biotin IgG in PBS is flowedacross the biotin region then hydrophobic region of the surface. Themicrofluidic channel is designed such that all anti-biotin IgG in thechannel is captured by the biotin-terminated region of the surface. Thesurface is imaged by using liquid crystals in a liquid crystal cell.Inspection of the hydrophobic region of the surface will reveal if thereare proteins in the sample other than the antibody (i.e., impurities).Thus this assay is useful for quality control. This experiment isrepeated with BSA doped into the anti-biotin IG. The presence of the BSAis detected by non-uniform orientation of the liquid crystal on thehydrophobic region of the surface.

Example 6

This Example describes the preparation of an anisotropic surface bynanoblasting. First, glass microscope slides are cleaned using piranasolution (H₂O₂/H₂SO₄). Caution must be used because this solution hasbeen reported to detonate upon contact with organic materials. Beads(50-1000 nm) are sprayed onto the surface of the glass microscope slidesusing a commercial abrasive spraying device (nanoblaster). A fixeddirection of incidence is used with an angle of incidence of 45 degrees,measured from the normal of the substrate. The surface of the glassslides is then functionalized by using aminopropyltrimethoxysilane. Thefunctionalized glass surface is activated by immersion into DSS. Theactivated glass microscope slide is immersed into an aqueous solution ofBSA. The surface is rinsed with PBS and then with water. Next, twomicroscope slides prepared as just described are assembled into anoptical cell. The slides are spaced apart by using a 20 micrometer-thickfilm of Mylar. The two slides are clipped together using bulldog clips.5CB is heated into its isotropic phase and wicked between the twosurfaces that define the optical cavity of the cell. The sample isallowed to cool to room temperature and then the optical cell isobserved in transmission using a polarized light microscope. When viewedbetween crossed polarizers, the sample will appear bright and dark(sequentially) when rotated.

Example 7

This Example describes the preparation of an anisotropic surface bynanoblasting. First, glass microscope slides are cleaned using piranasolution (H₂O₂/H₂SO₄). Caution must be used because this solution hasbeen reported to detonate upon contact with organic materials. Beads(50-100 nm) are sprayed onto the surface of the glass microscope slidesusing a commercial nanoblaster. A fixed direction of incidence is usedwith an angle of incidence of 45 degrees, measured from the normal ofthe substrate. The surface of the glass slides is then functionalized byusing aminopropyltrimethoxysilane. The functionalized glass surface isactivated by immersion into DSS. The activated glass microscope slide isimmersed into an aqueous solution of biotinylated BSA. The surface isrinsed with PBS and then with water. The glass microscope slidepresenting biotinylated BSA is then immersed into an aqueous solutioncontaining 1 micromolar of 100 nm-sized, streptavidin-coated beads. Thesurface is rinsed with PBS and then with water. Next, two microscopeslides prepared as just described are assembled into an optical cell.The slides are spaced apart by using a 20 micrometer-thick film ofMylar. The two slides are clipped together using bulldog clips. 5CB isheated into its isotropic phase and wicked between the two surfaces thatdefine the optical cavity of the cell. The sample is allowed to cool toroom temperature and then the optical cell is observed in transmissionusing a polarized light microscope. When viewed between crossedpolarizers, the sample appears non-uniform because the beads bound tothe surface will have erased the anisotropy introduced by the process ofnanoblasting.

Example 8

This Example describes the preparation of an anisotropic surface bynanoblasting. First, glass microscope slides are cleaned using pirhanasolution (H₂O₂/H₂SO₄). Caution must be used because this solution hasbeen reported to detonate upon contact with organic materials. Beads(50-100 nm) are sprayed onto the surface of the glass microscope slidesusing a commercial nanoblaster. A fixed direction of incidence is usedwith an angle of incidence of 45 degrees, measured from the normal ofthe substrate. Next, a gold film is obliquely deposited on the surfaceof the nanoblasted microscope slide at an angle of deposition of 50degrees (measured from the normal). An electron beam evaporator is usedto obliquely deposit 30 Angtroms of titanium and then 130 Angstroms ofgold onto immobile glass microscope slides. The rate of deposition ofthe metals is 0.2 Angstroms/sec. The film is deposited with an azimthualdirection of incidence that is parallel to that used to nanoblast thesurface. Biotinylated BSA is then adsorbed onto the surface of the gold.The protein-coated substrate is then immersed into an aqueous solutioncontaining 1 micromolar concentration of avidin-coated, 100 nm diameterbeads. Two microscope slides prepared as just described are thenassembled into an optical cell. The two surfaces are spaced apart byusing a 20 micrometer-thick film of Mylar and clipped together usingbulldog clips. Next, 5CB is heated into its isotropic phase and wickedbetween the two surfaces that define the optical cavity of the cell. Thesample is allowed to cool to room temperature and then observe theoptical cell in transmission using a polarized light microscope. Whenviewed between crossed polarizers, the sample appears non-uniform.

Example 9

This Example describes the preparation of an anisotropic surface bystretching a substrate. First, a sheet of polystyrene is heated aboveits glass transition temperature. A tensile stress is then applied bypulling at its two ends, and then the substrate is cooled below theglass transition temperature prior to releasing the tensile stress. Anoptical cell is then fabricated from the stretched polymer film and anOTS-coated glass microscope slide. OTS-coated glass microscope slidesare known to cause perpendicular (homeotropic alignment) of liquidcrystals. The two surfaces are spaced apart using 20 micrometer-thickfilm of Mylar. The cell is then mounted in a polarized light microscopewith an optical compensator. The compensator is adjusted to compensatefor any stress-induced birefringence in the polystyrene. Next, 5CB isheated into its isotropic phase and draw it into the optical cell byusing capillary action. The optical appearance of the 5CB is observedwithout further adjustment of the compensator. The 5CB appears uniformlydark or bright between crossed-polarized, indicating uniform alignmentof the liquid crystal on the stretched polymer surface.

Example 10

This Example describes the fabrication of heterogenous surfaces for usein LC assays. A one-millimeter-thick slab of PDMS is cast on the surfaceof a planar substrate. The PDMS is peeled from the surface of the planarsubstrate and wrapped around the surface of a cylinder with a diameterof 3 centimeters. The PDMS is held onto the cylinder by using rubberbands. Gold film is evaporated onto the surface of the PDMS wrappedaround the cylinder. An electron beam evaporator is used to obliquelydeposit 30 Angtroms of titanium and then 130 Angstroms of gold onto thePDMS. The rate of deposition of the metals is 0.2 Angstroms/sec. Thegold coated PDMS is released from the cylinder and mounted on thesurface of a glass microscope slide. A self-assembled monolayer ofhexadecanethiol is formed on the surface of the gold film by placing adroplet of a 1 mM ethanolic solution of PDMS onto the surface of thegold for 1 minute. The surface is rinsed with ethanol and then driedunder a stream of nitrogen. The gold coated PDMS is then assembled intoan optical cell using a second surface formed from OTS-coated glassmicroscope slide. The two surfaces forming the optical cell are spacedapart by 20 micrometers by using Mylar spacing material. 5CB is heatedinto its isotropic phase and wicked between the two surfaces bycapillary action. The optical appearance of the 5CB, once cooled to roomtemperature, is examined with a polarized light microscope using crossedpolars. The region of the surface onto which the gold is deposited atnormal or near-normal incidence causes non-uniform anchoring of theliquid crystal. That is, the liquid crystal in this region of thesurface is non-uniformly oriented. Away from this region, where the goldis deposited with an angle of incidence larger than 10 degrees, theanisotropy in the gold film causes the liquid crystal to assume auniform orientation. Thus, there is a gradient in the appearance of theliquid crystal (from non-uniform to uniform).

Example 11

This Example describes the fabrication of heterogenous surfaces for usein LC assays. A glass microscope slide is heated in a bunsen burner.When the glass is soft, the microscope slide is bent such that the twoplanar ends of it define an angle of 150 degrees (i.e., it is bent by 30degrees). The microscope slide looks like a “V”. The glass microscopeslide is cleaned in pirhana solution. The glass microscope slide is thenmounted in an electron beam evaporator, such that one surface of themicroscope slide is oriented at 30 degrees from the incident flux ofgold; and the second region of the microscope slide is oriented at anangle of 60 degrees from the incidence flux of gold.

The electron beam evaporator is used to obliquely deposit 30 Angtroms oftitanium and then 130 Angstroms of gold onto the PDMS. The rate ofdeposition of the metals is 0.2 Angstroms/sec. A self-assembledmonolayer is then formed from hexadecane thiol on the surface of thegold film by immersion into a 1 mM ethanolic solution ofhexadecanethiol. Next, BSA is adsorbed onto the hydrophobic SAMsupported on the surface of the gold film. Two pieces of OTS-coatedglass are then mounted on the gold film using 10 micrometer-thick Mylarspacers. The two cavities of the optical cell are filled with 5CB heatedinto its isotropic phase. The 5CB is then allowed to cool within thecavity. The appearance of the liquid crystal is observed using apolarized light microscope. On the region of the optical cell with goldfilm deposited at an angle of incidence of 30 degrees, the appearance ofthe liquid crystal is non-uniform. In contrast, in the region of theoptical cell with the gold film deposited at an angle of incidence of 60degrees, the liquid crystal appears uniform.

Example 12

This Example describes the fabrication of heterogenous surfaces for usein LC assays. Gold is obliquely deposited onto glass diffraction grating(blaze angle of 15 degree and a very long period) at a nominal angle ofincidence of 45 degrees from the normal of the grating. Due to the blazeangle, one surface of the grating will be coated with gold that depositsat 30 degrees whereas the other surface will be coated with goldincident at an angle of 60 degrees. A mixed monolayer is formed on thesurface of the gold film that presents biotin. Various samples areprepared that bind different amounts of anti-biotin IgG. Opticalmicroscopy is then used to record the optical appearance of the liquidcrystal as a function of the amount of bound anti-biotin IgG. Theoptical response of the liquid crystal on the grating surface iscompared to the optical response on a surface that is planar. On thegrating surface, the dynamic range of the response of the liquid crystalis larger.

Example 13

This Example describes LC assays prepared with a dichroic dye. A rubbedfilm of chemically immobilized biotinylated BSA is prepared. Next, adichroic dye (0.01%), such as azobenzene, is mixed into 5CB. The dye/5CBmixture is then heated into its isotropic phase. The rubbed film is usedto form an LC assay cell. The LC assay in then placed into a UV-Visspectrophotometer without a polarizer and a scan between 180 nm and 800nm is run for different orientations of the sample in thespectrophotometer. These steps are then repeated, except thatanti-biotin IgG is bound onto the surface of the rubbed film.Additionally, a parallel experiment is performed wherein a polarizingfilter is placed before the sample so that the sample is illuminatedwith polarized incident light. Whereas the absorbance spectrum of thecell prior to the binding of IgG is highly dependent on the orientationof the cell within the spectrophotometer, relatively little modulationin the intensity is seen when IgG is bound to the surface of the rubbedfilm of biotinylated BSA.

Example 14

This Example describes LC assays prepared with a fluorescent agent. Arubbed film of chemically immobilized biotinylated BSA is prepared.Next, an anisometric fluorescent dye (0.01%), such as BTBP, is mixedinto 5CB. The dye/5CB mixture is then heated into its isotropic phase.The rubbed film is used to form an LC assay cell. The LC assay in thenplaced into a fluorimeter (excitation at 488 nm) and the fluorescence at510-550 nm determined. These steps are then repeated, except thatanti-biotin IgG is bound onto the surface of the rubbed film.Additionally, a parallel experiment is performed wherein a polarizingfilter is placed before the sample so that the sample is illuminatedwith polarized incident light. Whereas the fluorescence from the cellprior to the binding of IgG is highly dependent on the orientation ofthe cell within the fluorimeter, relatively little modulation in theintensity is seen when IgG is bound to the surface of the rubbed film ofbiotinylated BSA.

Example 15

This Example describes the detection and quantification of bound analyteby measurement of the threshold electrical field required to change theorientation of the liquid crystal. First, a gold film is obliquelydeposited onto a glass microscope slide as described in detail above. Amixed, biotin presenting monolayer is then prepared on the gold film (asdescribed above). The slide is then half-dipped into an aqueous solutioncontaining anti-biotin IgG. An optical cell is then assembled from twosurfaces—one surface is the half-dipped sample and the second surface isa SAM formed from hexadecanethiol on gold. One end of the film is spacedapart using a ˜1 micrometer-thick spacer and space the other end of thecell with a 50 micrometer thick spacer. The cell is then filled with5CB. An AC electric field is then applied and, using a polarized lightmicroscope, the propagation of the reoriented liquid crystal across thewedge (the LC will reorient first at the thin end of the cell) isdetermined as a function of the magnitude of the applied voltage.

Example 16

This Example describes the detection and quantification of bound analyteby measurement of the threshold electrical field required to change theorientation of the liquid crystal. First, a gold film is obliquelydeposited onto a glass microscope slide as described in detail above. Amixed, biotin presenting monolayer is then prepared on the gold film (asdescribed above). The slide is then dipped into an aqueous solutioncontaining anto-biotin IgG. An optical cell is then assembled from twosurfaces—one surface is the half-dipped sample and the second surface isa SAM formed from hexadecanethiol on gold. The two surfaces are spacedapart by using a ˜5 micrometer-thick spacer. The cell is then filledwith 5CB. An AC electric field is then applied and, using a polarizedlight microscope, the threshold voltage required to observe or measure achange in orientation of the liquid crystal is determined.

Example 17

This example describes the detection of non-specific adsorption of BSAto a surface. An electron beam evaporator was used to obliquely depositapproximately 30 Angstroms of titanium and subsequently approximately300 Angstroms of gold onto an immobilized glass microscope slide. Therate of deposition of the metals was 0.2 Angstroms/sec and the angle ofdeposition was 69.5° from the normal. The gold substrate was thenimmersed into a 10 μM solution of bovine serum albumin (BSA) in PBS andincubated at room temperature for two hours. The BSA was then rinsedfrom the substrate using distilled, deionized water (ddH20) and driedunder a stream of nitrogen. The BSA-adsorbed gold slide was then pairedwith an octadecyltrichlorosilane (OTS)-treated glass slide that causedthe nematic phases of the 5CB liquid crystal to orient perpendicular tothe BSA-adsorbed gold slide. A thin film of Mylar (thickness 20 μm) wasused to separate the two surfaces, which was then secured using twosmall binder clips. Liquid crystal (5CB) was heated into its isotropicphase and introduced into the cavity between the two surfaces bycapillary forces. The entire liquid crystal cell was placed into apolarized light microscope and viewed between cross-polars (intransmission). The BSA that adsorbed onto the gold surface caused theliquid crystal to assume a non-uniform orientation, as depicted in FIG.3, whereas the surfaces that do not possess BSA non-specificallyadsorbed to the surface caused uniform alignment of the liquid crystal(FIG. 4). Thus, the liquid crystal was shown to be useful for theimaging of non-specific adsorption of a molecule to a surface.

Example 18

This example describes the preparation of an anisotropic surface bystretching a substrate. First, a small sheet of Parafilm was stretchedby pulling at the two opposing edges. It was then mounted on top of aglass microscope slide using two-sided tape at one edge of the glassslide to hold the Parafilm securely in place. An optical cell was thenfabricated from the stretched substrate and an OTS-coated glassmicroscope slide. OTS coated microscope slides are known to causeperpendicular (homeotropic alignment) of liquid crystals. The twosurfaces were spaced apart using a 50 μm thick film of Mylar. Liquidcrystal (5CB) is heated into its isotropic phase and placed on top ofthe substrate. The OTS slide was gently placed on top of the substrate,with care taken to avoid any air bubbles. Both non-stretched (FIG. 5)and stretched Parafilm (FIG. 6) were analyzed. The 5CB liquid crystalappears uniformly dark or bright between crossed-polarizers, indicatinguniform alignment of the liquid crystal on the stretched polymersurface. These results demonstrate that the Parafilm was alreadystretched in the manufacturing process, and thus additional stretchingdid not affect the homeotropic alignment when coupled with an OTS-coatedslide.

Example 19

This Example describes a second experiment that further described thepreparation of an anisotropic surface by stretching a substrate. A smallstrip of Parafilm was incubated in 10 mg/mL BSA in PBS at roomtemperature for two hours. The BSA was rinsed off of the glassmicroscope slide using ddH20 and dried under a stream of nitrogen. Theliquid crystal cell was fabricated and loaded in same fashion asdescribed above in Example 18. FIGS. 7 and 8 show that BSA was able toadsorb into both the stretched and non-stretched Parafilm, as indicatedby the pronounced color modulation upon rotation (undercrossed-polarizers) by 45°. Modulation was also observed in thestretched Parafilm indicating homeotropic alignment.

Example 20

This Example describes liquid crystal assays prepared with azobenzene, adichroic dye. One cell was fabricated from two OTS-coated glassmicroscope slides, which produces homeotropic alignment of the liquidcrystal, and a second cell was fabricated by pairing a rubbed BSA (0.1mg/ml) glass microscope slide together with a regular glass microscopeslide, which produces planar alignment of the liquid crystal. Next, theazobenzene dye was mixed into the 5CB liquid crystal. The dye/5CBmixture was then heated into its isotropic phase and injected into eachof the cells via capillary action. The liquid crystal cell was thenmounted into a UV-VIS spectrophotometer (Shimadzu Bio-Spec 1601,Shimadzu, Kyoto, Japan) and a scan between 190 nm and 800 nm was run foreach of the different orientations. The results showed an absorbancepeak at ˜447 nm which was more intense for the planar orientation of theliquid crystal (0.362) than the homeotropic orientation (0.200). Thus,absorbance readings were able to distinguish between the variousorientations of the liquid crystal.

Example 21

This Example describes an example similar to that described in Example20 above. In this case, BTBP(N,N′-Bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenedicarboximide) wasused. This example describes liquid crystal assays prepared with BTBP.One cell was fabricated to give homeotropic alignment of the liquidcrystal and one cell to give planar liquid crystal alignment (seeExample 20). Next, BTBP dye was mixed into the 5CB liquid crystal. Thedye/5CB mixture was then heated into its isotropic phase and injectedinto each of the cells via capillary action. The liquid crystal cell wasthen mounted into a UV-VIS spectrophotometer (Shimadzu Bio-Spec 1601)and a scan between 190 nm and 800 nm was run for each of the differentorientations. The results showed three absorbance peaks at ˜532 nm, 495nm, and 464 nm. As observed in Example 21, the absorbance values weremore intense for the planar orientation (0.364, 0.308, 0.224respectively) as compared to 0.250, 0.193, and 0.128 for the homeotropicorientation. Thus, initial absorbance readings were able to distinguishbetween the various orientations of the liquid crystal.

Example 22

This example describes the detection and quantification of a boundanalyte by measurement of the threshold electrical field required tochange the orientation of the liquid crystal. First, a gold film wasobliquely deposited onto a glass microscope slide. An electron beamevaporator was used to obliquely deposit ˜30 Angstroms of titanium andthen ˜300 Angstroms of gold onto an immobilized glass microscope slide.The rate of deposition of the metals was 0.2 Angstroms/sec and the angleof deposition was 30° from the normal. The slide was then half-dippedinto an aqueous solution of 0.1 mg/ml BSA and allowed to incubate for atroom temperature for two hours. The BSA was then rinsed from the glassslide with ddH₂O and dried under a stream of nitrogen. An optical cellwas then assembled from two surfaces—one surface was the half-dipped BSAglass microscope slide and the second surface was a regularobliquely-coated (30°) gold microscope slide. The two surfaces werespaced using ˜4 μm Saran Wrap. The cell was then filled with liquidcrystal (5CB). A DC electric field is applied and, using a polarizedlight microscope, the threshold voltage required to observe or measure achange in orientation of the liquid crystal was determined. A voltage of˜6.5 V was necessary to see the beginnings of an orientational liquidcrystal change. The untreated gold portion of the glass microscope slideappeared to change first, whereas the BSA-treated half started to changeshortly thereafter. Upon ramping the voltage to ˜15 V, the entire sampleshowed homeotropic liquid crystal alignment (as indicated by a“black-cross” when viewed with a Bertrand microscope lens.) Thus, theapplication of an electrical field allows for the detection of a boundprotein by altering the orientation of liquid crystal.

Example 23

This example describes the preparation of an anisotropic surface bytexturizing a substrate (nanoblasting). First, glass microscope slideswere cleaned using an LF-5 Plasma Asher (Mercator Control Systems, Inc).The glass microscope slides were then texturized by rubbing a fine320-grit sandpaper pad (3M, St. Paul, Minn.) across the surface of theslide in a uniform direction approximately five times (keeping thepressure fairly constant). The rubbing distance was approximatelythirteen centimeters. An optical cell was then fabricated from thetextured glass slide and a clean glass microscope slide, with a 20 μmMylar spacer placed in between. Liquid crystal (5CB) was heated into itsisotropic phase and injected between the two surfaces via capillaryaction. The cell was allowed to cool and then observed under apolarizing microscope.

The results are shown in FIG. 7. When viewed between cross polarizers,the cell appears bright and dark (sequentially) when rotated. Thus, theanisostropic surface prepared by roughening the substrate resulted in auniform alignment of the liquid crystal.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in organicchemistry, materials science, chemical engineering, virology, biology,genetics, or related fields are intended to be within the scope of thefollowing claims.

1. A method comprising: a. providing a sample suspected of containing ananalyte and a liquid crystal assay device; b. adding said analyte tosaid liquid crystal assay device under conditions such that the presenceof said analyte causes a detectable ordering of mesogens in said liquidcrystal assay device; and c. quantitating the amount of said analyte insaid sample based on said detectable ordering of mesogens.
 2. The methodof claim 1 wherein said liquid crystal assay device comprises: a. afirst substrate having a surface, said surface comprising a recognitionmoiety; and b. a mesogenic layer oriented on said surface.
 3. The methodof claim 2, wherein said liquid crystal assay device further comprisesan interface between said mesogenic layer and a member selected from thegroup consisting of gases, liquids, solids, and combinations thereof. 4.The method of claim 2, wherein said recognition moiety is attached tosaid surface by an interaction which is a member selected from the groupconsisting of covalent bonding, ionic bonding, chemisorption,physisorption, and combinations thereof.
 5. The method of claim 2,wherein said surface further comprises an organic layer.
 6. The methodof claim 5, wherein said recognition moiety is attached to said organiclayer by an interaction which is a member selected from the groupconsisting of covalent bonding, ionic bonding, chemisorption,physisorption, and combinations thereof.
 7. The method of claim 2,wherein said mesogenic layer comprises a polymeric mesogen.
 8. Themethod of claim 1, wherein said mesogen is selected from the groupconsisting of 4-cyano-4′-pentylbiphenyl,N-(4-methoxybenzylidene)-4-butlyaniline and combinations thereof.
 9. Themethod of claim 1, wherein said mesogenic layer comprises a lyotropicliquid crystal.
 10. The method of claim 2, wherein said surface is ametal surface.
 11. The method of claim 10, wherein said metal surface isselected from the group consisting of gold, platinum, palladium, copper,nickel, silver, and combinations thereof.
 12. The method of claim 2,wherein said substrate is selected from the group consisting of flexiblesubstrates, rigid substrates, optically opaque substrates, opticallytransparent substrates, conducting substrates, semiconductingsubstrates, and combinations thereof.
 13. The method of claim 2, whereinsaid substrate is selected from the group consisting of inorganiccrystals, inorganic glasses, inorganic oxides, metals, organic polymers,and combinations thereof.
 14. The method of claim 13, herein saidorganic polymer is selected from the group consisting of polyvinylidenefluoride, polydimethylsiloxane, polycarbonate, polystyrene,polyurethane, polyisocyanoacrylate, epoxy and combinations thereof. 15.The method of claim 2, wherein said substrate is heterogenous.
 16. Themethod of claim 15, wherein said heterogeneity is a gradient oftopography across the said surface.
 17. The method of claim 15, whereindifference in liquid crystal orientation across said gradient oftopography is correlated to the concentration of said analyte in saidsample.
 18. The method of claim 2, wherein said device further comprisesa dichroic dye in said mesogenic layer.
 19. The method of claim 18,further comprising the step of measuring the amount of light transmittedby said device, wherein the amount of light transmitted is proportionalto the amount of said analyte in said sample.
 20. The method of claim 2,wherein said device further comprises a dichroic agent in said mesogeniclayer.
 21. The method of claim 1, wherein said quantitating stepcomprises illuminating said liquid crystal assay device with a specificwavelength of light to determine the degree of disorder introduced intosaid liquid crystal assay device.
 22. The method of claim 21, furthercomprising the step of measuring the amount of light transmitted by saiddevice, wherein the amount of light transmitted is proportional to theamount of said analyte in said sample.
 23. The method of claim 1,wherein said quantitating step is performed with a plate reader.
 24. Themethod of claim 23, wherein said plate reader is utilized to detect saiddetectable ordering of mesogens, wherein said detectable ordering ofmesogens is accompanied by a change selected from the group theconsisting of a change in fluorescence, transmittance, birefringence,and absorbance changes that accompany the reorientation of the liquidcrystal.
 25. The method of claim 1, wherein said quantitating step isperformed by measurement of the threshold electrical field required tochange said ordering of said mesogens.
 26. The method of claim 2,wherein said liquid crystal assay device further comprises electrodes,wherein said electrodes apply an electric field across said device. 27.The method of claim 1, wherein said analyte is selected from the groupconsisting of polypeptides, polynucleotides, organic analytes, andpathogens.